Competent Cells - Science topic

I am using a vector that has repeat sequences, and using Mach1 competent cells. I heard that if the cells are not stable cells, then we shouldn't grow them in 37 to avoid recombination to happen between the repeat sequences.

I would like to transfect bacteria with some plasmids. However, the bacteria are not competent but either isolated from the environment or purchased reference strains.

Can I transfect these strains?

Is there a kit or an established protocol?

Is the procedure much more different or the efficiency much lower than with competent cells?

Thank you

Hello everyone!

I transformed my vector PL4440 containing the gene of interest, into HT115 competent cells. However the culrure can not grow on LB agar plates (amp + tetr ) antibiotic. Culture can grow from liquid to liquid LB containing Ampicilin 50 mg/ml and tetracycline 12,5 mg/ml. Had anyone this experience ?

Hello all. I transformed some Agrobacterium competent cells by electroporation and performed colony PCR to see the positive transformants. However, I performed the glycerol stock after 10 days of streaking the cells in a new plate for colony PCR. This plate was stored at -4 degrees. I know that I should make the glycerol stock as soon as I confirmed the transformants, but do you think my cells lost viability during 10 days in the plate before going to -80°C? Thanks

I have been working with E. coli DH5 alpha competent cells to amplify my PLJM1 plasmid with an insert, but it just doesn't get amplified in the appropriate amount! The bacterial yield is too low as well. But the same plasmid with ERK-2-insert gets amplified in the same bacteria. The bacteria also seem to multiply plasmids very well in the lab next to ours. I have tried the amplification many times, and it's still just a very faint band in the gel. Totally clueless as to why this is happening when the same bacteria with the same plasmid but a different insert grows in the same media, but this doesn't.

I have been trying insert my reporter construct (1.8kb - taken from another plasmid) into my lentiviral vector (9.6kb). They have compatible restriction sites - NheI and EcoRI. I digested the plasmid and insert with the enzymes; gel purified my vector backbone and insert and set up the ligation reaction.

Result of the plates are:

A - ligation reaction - approx 25 colonies. (used 20ul of the ligated mix)

B - vector backbone only - approx 10 colonies

C - insert only - approx 10 colonies

D - no ligase - several colonies

E - EcoRI linearized vector - several colonies

F - EcoRI linearized plasmid containing the insert - no colonies

G - only competent cells - no colonies

I don't understand why i have colonies in my insert only plate. the insert doesn't even have Amp resistance gene. Could there have been some contamination with the vector backbone? Why do i have colonies in the vector backbone only control? does the T4 ligase also ligate uncomplimentary ends in a plasmid??

I have cloned a 6.46 kb having restriction sites for enzymes BamHI and Not I in pcDNA3.1 vector. After colony pcr screening, the result shows positive result with primer specific to vector and insert region. However no insert size was detected by digestion. Note. ligation reaction was transformed into DH5a competent cells. I am not sure whether DH5a is suitable for large insert size. Thank you so much for kind suggestion.

A plasmid of mine has GOI in opposite orientation. I have another plasmid which has exact same RE site but in such a way that it orients my GOI in right direction. However even after multiple attempts of RE digestion, Gel extraction followed by ligation, I didn't got any colonies on my transformation plate. I also checked efficiency of my competent cell with empty vector and found no issue (got 200-300 colonies) . I would be grateful if anyone can suggest me any possible solution.

Hello. I'm writing with courage to seek advice from professors and researchers. Currently, I am researching the impact of introducing a B plasmid into E. coli carrying an A plasmid, to study the effect of B genes on A genes.

After transforming the A plasmid (ampicillin resistance) via electroporation, I culture the cells to create electroporation competent cells, and then perform electroporation transformation with the B plasmid (gentamicin resistance).

While transforming A and B separately into DH10B Competent cells works well, colony formation does not occur when transforming B into A-harboring competent cells.

A plasmid: ColE1 origin, B plasmid: oriV, so there should be no incompatibility issues.

I wonder if adding ampicillin (1X, 100mg/ml) during the culturing process after transforming A could affect the cells. I tried dividing the cultures into small cultures, always adding 1x ampicillin, and when doing large cultures, I tried not adding antibiotics, or adding them at 0.2x concentration, but in all cases, transformation hardly occurs.

Should I consider anything else? Could co-transformation be the solution?

I would greatly appreciate your help. Please let me know if you need more information for your response.

I was making competent cells the other day with a culture volume of 100 mL (diluted from an O/N) in a 500 mL flask.

Starting at 1 h, I started measuring OD to check when the cultures reached OD600 of 0.4. I use a BioWave CO8000 cell density meter. I take 500 uL of culture and measure it against a blank of the medium.

But in the time I take it to the bench and then remove 500 uL, is that measurement really going to be accurate? Is there a better way to monitor the OD of a large culture? Would I be able to set up a smaller culture in a 15 mL tube at a similar dilution such that I can just take the reading from the culture tube? Please advise. Thank you!

I have prepared competent cells of L. lactis NZ9000. But transformation not working. No colony at all in the plate. Parameters are as following:

plasmid concentration: 1 uL (300ng)

Electroporator- Voltage 2.0, 1 pulse

Cells are viable (checked), Antibiotic conc. 10 ug/mL in plate

100 uL spread in one plate, Spin down...then rest of the cells spread in another plate.....no colonies.

Protocol followed:

1. refresh (test tube): 5 mL of GM17 liquid medium was inoculated with 1% L. lactis NZ9000 from glycerol stock and incubated at 30oC for 24 hours.

2. pre-culture (test tube): Two 5 mL bottles of GM17 liquid medium were inoculated with 1% of the refreshed culture and incubated at 30oC for 24 hours.

3. main culture (reagent bottle):

One thousand mL of SGM17 medium (42.5 g/L M17 broth, 102.7 g/L Sucrose, 10 g/L Glycine, 5 g/L Glucose) was inoculated with 1% of the pre-culture medium and incubated at 30oC until OD600 = 0.5 to 0.6.

4. the main culture was ice-cooled for 5 min.

5. The culture was transferred to four ice-cooled 500 mL centrifuge tubes and centrifuged at 4oC for 25 minutes at 5,480 x g. The bacteria were collected.

6. 40 mL of washing solution (10% (v/v) Glycerol, 136.9 g/L Sucrose) was added to each centrifuge tube and resuspended.

7. Each resuspension was transferred to ice-cooled 50 mL Falcon tubes, centrifuged at 4oC, 2,300 x g for 15 min, and the bacteria were collected.

8. 40 mL of washing solution was added to two of the four Falcon tubes and resuspended, then each was transferred to the other two tubes and resuspended again.

9. the bacteria were collected by centrifugation as in step 7.

10. 40 mL of washing solution was added to each of the two Falcon tubes and resuspended.

11. Centrifuged as in step 7 and collected the bacteria.

12. add 1.2 mL of wash solution to each of the two Falcon tubes and resuspend.

13. The resuspension solution was dispensed in 50 µL portions into PCR tubes arranged in a cooled PCR tube stand to make NZ9000 competent cells. After preparation, the cells were stored at -80oC.

Two plasmids of different sizes were constructed based on the pBeloBAC11 backbone. One is 18kd and the other is 40kd. Choose to use TOP10 competent cells and transform in CaCl2 solution. The 18KD can be transformed successfully, but the 40kd size does not get colonies. How to optimize My method to transform a 40kd plasmid? Does the method of power transfer help?

I have a plasmid with kanamycine antibiotic resistant gene and Bar as a marker gene. I need to transfer this plasmid into AGL-1 strain of agrobacterium tumefaciens. I faced a problem when I follow the protocol steps of transformation. I did not get bacterial colony even after 2-days of a LB media plate having Kan 50ug/ml.

Protocol steps

1- Take competent cells from -80 C.

2- Add 5ul of plasmid having conc. 50ng/ul in 100 ul of AGL-1 BACTERIA.

3- Keep on ice for 30 min.

4- put in liquid nitrogen for 5 min

5- keep on heat bath for 5 min.

6- keep on ice for 5-min again.

7- add 800ul of LB without antibiotic (Kan)

8- Shake for 2-hrs at 28 C.

9- spread on LB media plate with kan 50ug/ml. Keep these plates on 28 C for 2-days.

But did not get the bacterial colony.

These are the protocol steps which I followed. Anyone can guide me where I am doing mistake?

Hello,

I purchased competent Rosetta2 cells (a BL21 expression strain of E. coli) and my transformations yielded 30-50+ CFUs per SOC plate. However, when I transfer the colonies to liquid culture, there is no growth after 20 hours.

It is a dual antibiotic system, chloramphenicol (100ug/mL) and kanamycin (50ug/mL). I've tried growing the colonies in SOC and LB. However, I am using colonies that I kept at 4C for 72 hours - could this be a problem?

I notice that when I inspect the colonies after refrigeration, there are these crystal growths (my PI believes they are phosphate crystals). Could micro-crystals be forming in my colonies, killing them?

All of my stocks have been autoclaved at 121C for 30 minutes and no one else in lab is having growth issues. I am using filtered tips. Note I am using an expression plasmid that's been successfully used in lab for many years/projects.

My next approach is to try restreaking a colony/repeating the transformation and trying only fresh colonies that haven't been refrigerated. Negative control plates (non-transformed competent cells) don't have any CFUs. Everyone is puzzled at my growth challenges.

Best,

Dan

I have inserted crispr vector in BL21 competent cell with ampicillin resistance and after induction with IPTG for protein ...no cas protein band was obtained using SDS page.. not even near to its size both in the supernantant as well as in the pellet... Can anyone help me.. the vector I ordered contains 6x his tag... But no protein after NI Nta agarose...

I am working with plasmids containing aion channel, with the goal of eventually using them for transfection in Hek cells.  The problem I am having is preparing these plasmids at the bacterial transformation stage.  I have 2 different channels (both from he HCN family), on of them (HCN2) grew perfectly on the first try in XL1 Blue cells.  However I am now doing point mutations on the channel (using a Quikchange kit) and I cannot get a colony that has my intact channel.  Additionally I am trying to use HCN1, another member of the same family, and it is giving me similar problems to the mutation reaction.  Here is what I have tried so far:

1.  I am using internal channel specific primers to screen picked colonies for the presence of my plasmid.  PCR of the unmated HCN2 plasmid produce a clean band of the appropriate size.  PCR of the mutation reaction prior to transformation produces a single band of the right size. but PCR's of the picked colonies for the mutants do not, they show multiple bands.

2. Used Stbl2 competent cell to hopefully prevent recombination of the plasmid,but the pct's looked the same as the XL1-Blue.  

3. Tried incubation at 37 and 30 degrees, and decreasing the antibiotic concentration, but still the same problem

I have tried these things with both the HCN2 mutation reaction and the wild type HCN1 plasmid and have had no luck.

Any advice would be much appreciated!  Also, if there are any extra details that would help please let me know 

Thanks! 

Anna

I have to clone a 1920 bp insert cut with NheI and HIndIII into my 7767bp vector cut with the same enzymes. Protocol used:

1. I cut 5 ug of insert vector and 2 ug of destination vector and gel purified it.

2. Performed ligation at 1:1, 1:3 and 1:5 ratios as a 10 ul reaction using T4 ligase from NEB at 16 C overnight.

3. Transformed 5 ul of ligation mix into NEB5 alpha, XL-10 Gold ultracompetent and Thermo DH5alpha competent cells according to manufacture's protocol and plated on LB +Amp plates with incubation overnight.

All reagents are new. However, I don't see any colonies after transformation. My gel picture shows that ligation has occurred I think. Lane 5, 6 and 7 are ligation products at 1:1, 1:3, and 1:5 ratios respectively. Lane 3 is cut insert and lane 10 is cut destination vector.

What could be going wrong?

Currently, I'm working on cloning experiments using competent cells (E.coli--> DH5-alpha). I've performed ligation before and continued with transformation in competent cells. after incubation overnight, I got colonies, then I spread the colony in Amp+X-gal+IPT to perform blue-white screening. unfortunately, after 16 hours in 37 degrees condition, I checked, and I didn't found any colonies grown. i did twice and the results were the same. could anyone know what caused my colony not to grow at all? thank you for your answer.

I am currently doing my PhD project which consists of a lot of cloning of new plasmids I am assembling. Our laboratory generally maintains the collection on JM109 strain. But since I am doing a lot of Gibson Assemblies, I have been using electrocompetent DH10B cells for higher efficiency. My question is, can I use standard protocol of preparation of electrocompetent E. coli on JM109 instead of DH10B?

I am trying to transform DH5-alpha E. coli with plasmids containing a desired gene along with the amp resistance gene. I have done this exact procedure several times before with the same plasmid (containing different desired genes) with no issue, but this time, I am getting no colonies at all. My competent cells seem to be fine as the cell aliquot transformed with my positive control plasmid grew fine.

My protocol involves incubating 20 uL aliquots of competent cells with 10 ng of DNA for 30 minutes on ice, then heat shock at 42.5 degrees C for 40 seconds followed by incubating on ice for 2 minutes. Then, I add 250 uL of pre-warmed LB broth without antibiotics and incubate in a shaker incubator at 37 degrees C for 1 hour. Then, I plate 100 uL of the mixture on plates containing ampicillin and let the plates incubate overnight at 37 degrees C.

Any suggestions would be appreciated.

Hi all.

For my last LR reaction I used SURE competent cells cells (link) for the transformation (since the final plasmid was very big).

I got many colonies (which is always suspicious) and end up finding that most (if not all) had the pdest vector (wich has the CCDB lethal gene). I finally transform 3 different destination vectors in SURE cells and DH5alpha and got to the conclusion that SURE cells seems to be resistant to the ccdb gene.

This resistance is not reported by the company. Does anyone has experienced with this type of competent cells? Is it normal this resistance?

Thanks

Hi all,

Hope you are having a nice day! I was wondering if anyone has used the bac-to-bac baculovirus expression kit to express recombinant protein? There is a step when creating the virus, where you transform a shuttle vector into DH10bac competent cells which contain a bacmid. This recombines with the shuttle vector to introduce your gene of interest into the bacmid, which you then isolate. I have followed this procedure using the manufacturer's specifications, and do get colonies. When I screen my control (uses a control vector sent with kit), the recombination did occur, but did not occur with my gene. I picked six colonies from each. The control vector has an insert which is a similar size to my insert. You select with gentamicin (shuttle vector encodes resistance), tetracycline (helper plasmid which contains genes that help facilitate the recombination event), and kanamycin (bacmid has resistance gene). Blue/white selection with bluo-gal and IPTG is supposed to select for recombination, colonies with recombinant bacmid should be white, not blue. I bought new bluo gal for my plates, so definitely should be good, and am getting many colonies on my plate but none are blue (which again would be negative.) However, when I screen them, they contained unrecombined bacmid. I have just picked 20 colonies off of the plate and am screening with colony PCR. My next ideas if I don't get anything from that are to up the bluo gal, up the IPTG, and troubleshoot/ use more of my shuttle vector in case actual transformation is the problem (kit has me using 1 ng which to me, seems low for this application.) The cells are expensive however, and I don't know much about optimizing recombination/ratios, etc. Does anyone have any suggestions? Sorry for any incorrect terminology or statements, I am completely new to this system. Also, I have attached a schematic of the system from the manual in case this helps.

Thanks,

Claire

Also, sizes:

Shuttle vector + my gene: approx 8 kb

Bacmid: 135 kb

I am using a pET28B vector to clone my 2.1kb insert digested with BamH1 (fwd) and Xho1 (rev) restriction enzymes. I'm using over 1ug of DNA for restriction digestion, kept at 37°C for over 3 hours. For cohesive end ligation, I'm using Promega T4 ligase, and keeping the setup (3:1, 5:1 and 7:1; insert: vector) for 16 hours before transforming in Dh5alpha competent cells followed by plating on Kan plates. I've been doing this for quite sometime now. But everytime, all I get are false colonies. I've tried to troubleshoot every single step but to no avail. My ligase is also new.

Hi all,

I've never worked with E.coli strains that already carry a plasmid and tried to make competent cells from them.. is it simply the usual protocol for chemically competent cells, but add the appropriate antibiotic in each step?

Your advice will be much appreciated

Cheers

Lisa

Hello,

I am attempting to co-express two different genes in Pichia pastoris X-33. My colleague successfully achieved this by transforming two different vectors simultaneously. Essentially, he added two distinct plasmids to the competent cells and performed electroporation. However, I am struggling to replicate this efficiently without resorting to multiple attempts and relying on luck. Do you have any suggestions on how to enhance the efficiency of this process?

One idea was to transform one gene and then the other one, with each vector containing a different antibiotic resistance. However, this strain is known to use only Zeocin (correct me if I am wrong).

Another obvious answer would be to create a construct with two different genes. However, in my work, I need to use any different gene combination. So, this would add too much extra work.

Thank you, and I hope you're all having a lovely New Year.

We had to put our pJet1.2 plasmid in a XL1-Blue Competent Cells. The result were very small colonies on a LB agar medium with ampicilin and IPTG. bands with a very low concentration were visible after gel electrophoresis. Is there a different bacteria that we can use that can produce more plasmids so we can see it with gel electrophoresis?

We have recently purchased competent shuffle E. coli cells from NEB, and we have to prepare more chemically competent cells, but I don't know if it would be better to grow the starter culture and the culture in the growth phase at 30ºC or 37ºC.

Any help would be useful, thanks!

Dear biology scientists,

Hello, I should do transduct lentivirus in the 293FT (HEK293) cells soon.

However, I have failed tranformation because the DH5a colonies were too low.

Let me tell my experimental procedure as below.

1. Making 10 mL of LB agar plate without any antibiotics

2. Spreading 10 mg/mL of Ampicillin in the 10 mL of LB agar plate

3. Melting the Escherichia coli DH5a in the ice

4. Aliquoting the DH5a by 30 uL per each sample

5. Inserting a vertor sample in the DH5a by doing spiral pipetting

6. 30 min incubation in the ice

7. 42 degree Celcius for heat-shock

8. 2 min incubation in the ice

9. Putting 1 mL of LB broth without any antibiotics into the heat-shocked DH5a

10. 37 degree Celcius incubation in a shaker for 45 min

11. 13000 rpm, 2 min, room temperature centrifuge

12. Spreading 100 uL of supernatant in the LB agar plate with 100 ug/mL Ampicillin

13. 37 degree Celcius incubation in an incubation

I have no idea why my colonies were rarely shown.

Another person did my procedure, and she got many pMD2G colonies and psPAX2 colonies.

What is my problem? Please help me.

Hello I have been trying to transform pet28b plasmids into Rosetta cells. I have tried two plasmids that already show expression in BL21 ( DE3) cells. Is there a list of compatible plasmids that can only be transformed into Rosetta cells? Also, does the factor that Rosetta cells have a resistant gene to Cloramphenicol has something to do? because my pet28b plasmid has a resistant gene to Kanamycin

I have purchased this plasmid from addgene:

From the agar stab, I have been able to grow the plasmid on LB-ampicillin plates (100mg/L) and it grows great. I isolated the plasmid using a miniprep kit, and it is exactly where it should be on a gel.

Then I tried to transform the plasmid into BL21(DE3) competent cells (NEB). No luck.

I have been troubleshooting this for months. I use pUC19 as a positive transformation control, and it always works really efficiently. I have tried changing DNA concentration, volume, heat shock time, recovery time, everything I can think of. I cannot get this plasmid into cells. Please help!

I prepared electrocompetents of the AGL1 strain following the "water" protocol: the bacterial culture was resuspended in water, subjected to two washes in water, one in 10% glycerol, and finally resuspended in 10% glycerol. When I attempt to electroporate, arcing occurs. This happens even when I use only the competent cells without the plasmid, indicating that it is not due to the presence of salts in the miniprep.

I prepared electrocompetents of the AGL1 strain following the "water" protocol: the bacterial culture was resuspended in water, subjected to two washes in water, one in 10% glycerol, and finally resuspended in 10% glycerol. When I attempt to electroporate, arcing occurs. This happens even when I use only the competent cells without the plasmid, indicating that it is not due to the presence of salts in the miniprep.

I have a 22kb backbone that needs to use Fse1 (NEB) and Kfl1 (Thermo) to cut to(21+kb and 700+bp bands). Still, the 2 enzymes come from different companies and their buffers are not the same(rcut-smart vs. fastdigest10 buffer), No matter whether I digested them together or separately, I couldn't get the right band, so I guess Fse1 didn't play a role ( I have already did the dam–/dcm– Competent E. coli C2925 transformation firstly), but Fse1 still can not cut my plasmid... So what should I do, try HST04 dam-/dcmCompetent Cells (form TAKARA)??? or redesign the vector?

I am doing Pcr cloning trying to clone a 3000 bp pcr product into a 9000 bp pcdh_bec1 plasmid .digestion was done using Xba1 and Not1 ,after gel extraction loaded 2ul on the gel and observed faint band of backbone. for ligation used 2ul(10ng/ul) backbone and 17ul of the insert(24ng/ul). didn't get any colonies I am using stbl2 cells growing them at 30 degrees to reduce chances of recombination.

when i followed the same strategy with the Plvx vector using the same competent cells i observed good amount of colonies although efficiency of competent cells was low.

Can someone help?

I have JM109 competent cell from PROMEGA.As the stock is about to finish, iam planning to make more competent cells following usual CaCl2 mediated protocol.My question is whether JM109 that is with me can be used as strain to proceed with the plan or else i need to procure one mutant strain? If i need to procure the strain, then where to in India?

Hi everyone

I have 2 vectors and I want to transform these into one bacteria cell (SHuffle strain).

Do you think co-transformation (Transforming 2 vectors together in one step into the competent cell) has an effect on Protein expression?

Or, that I have to transform the vectors step by step (e.g. transforming vector number 1 after that competent this cell and transform vector number 2).

is really different between these two strategies for Protein expression?

Note: I used co-transformation for these 2 vectors But the Expression rate was so low Compared with when I Expressed one vector Separately.

I did yeast two hybrid transformation a few days ago and no yeast colonies grow in the DDO+ 225 AbA plates after 3-5 days. OD600, carrier DNA, competent cell, as well as the amount of AD and BD plasmid were correct according to TAKARA manual. Can anyone know the reason why no colonies could grow in the plate?

I transformed a library into stbl3 competent cells following a standard protocol which is like incubation 30min in ice, 42°C for 45s, soc culture at 37°C for 1h, before culturing at 30°C on LB Amp+ dishes.

some supplementary info:

1. The dishes look normal after 30°C culture.

2. The plasmid was tested and had no problem.

3. I used another plasmid to tansform and harvested bacteria directly from the dishes(didn't frozen in refrigerator) with correct plasmid.

I can only find one reason for this phenomenon is that -80 made it happened. Can you help me find other factors?

Hello,

I am having difficulties working with E. coli WK6 cells.

I have used the WK6 cells to express Nanobodies for almost three years, and everything has gone well. One difficulty I encountered was preparing a fresh competent cells every month, unlike other E. coli strains in our lab.

The second difficulty I am facing is that I've tried six times over the past month to create a new batch of cells. Despite trying different approaches, I've been unable to get competent cells that are able to preform.

My protocol for wk6 preparation is based on Inoue et al.'s (1990) Gene 96:23-28.

I would appreciate any help you can offer.

I have transformed around 1000 ng of my circular plasmid into E. coli BL21 (DE3). However, there was no colony but a transparent bubble-like 'colony' formed at the bottom of the plate (As attached) . May I know what is it and how to overcome this?

I think there is nothing wrong with my competent cells as the transformation of positive control (blank vector) was successful.

I am currently making bacmid and consequent baculovirus using pDEST8, pFastBac and 438a vectors and recently switched to DH10EmBacY cells instead of DH10Bac due to the added YFP signal to monitor transfection, etc.

I was wondering if there is any reason for me to not use either of the two competent cells interchangeably to make bacmid DNA?

I am using Escherichia coli MC1064 as competent cells to insert an empty vector. I use CD 3-434 Vector(https://www.arabidopsis.org/servlets/TairObject?type=stock&id=313445) . i used kanamycin as antibiotic and then i realized this is for screening in plant . i haven't any map of it. does anyone know what antibiotic should i use?

i was cloned the lysin gene of bacteriophage in the pGEX 4t1 vector and transformed them in the BL21 Competent cell. competent cell was transformed and upon induction they are showing the induction of desired band of recombinant protein size in the sds page. but when for their purification purpose i was breaking the cell by using three times freeze thaw cycle at -80degree Celsius. after freeze thaw cycle i observed that my lysate become two viscous that it cannot got separated into supernant and pellet after centrifugation at 13000 rpm for 20minutes

please give me the reason why this happen with my clone?

optimized conditions for competent cell preparation

I made some competent cells, but using those cells for molecular cloning will produce a lot of wrong plasmids, and the colonies grow very few, so will competent cells affect molecular cloning to construct plasmids?

I have been working on site-directed mutagenesis for a few months now, without success. I used a High-Fidelity DNA polymerase to amplify my vector with an insert and saw the target band after the PCR when I ran the product on a gel. I added 0.5µl DpnI to my 50 µL PCR reaction, and incubate at 37°C for an hour. Then, I used 25ul of the PCR product for transformation to 100µL competent XL1-blue cells. After overnight incubation at 37°C, I had got no mutant colonies. The positive control had a lot of colonies and the negative control (without transformation) had no colony, so I think my competent cells work well.

Additionally, I had previously done this work and had managed to get colonies but this time around I am not getting anything at all.

Anyone with expertise on this, may I please get some assistance? Thank you so much.

I am working with two plasmids that both call for Stabl3 competent cells for growth in bacteria, how can I find out if these plasmids would grow in NEB 10 competent cells? They did not transform with DH5a.

I used the following procedure to transform DH10Bac E. coli.

I wanted to know why my experiment failed.

1. Thaw 25ul DH10Bac competent cells on the ice for each transformation.

2. Add pFastBac™ construct: 1 ng (1 μL) plasmid DNA to the DH10Ba competent cells and mix gently.

3. Incubate cells on ice for 30 minutes.

4. Heat-shock the cells for 45 seconds at 70°C without shaking.

5. Immediately transfer the tubes to ice and chill for 3 minutes.

6. Add 900 μL of room temperature LB.

7. Shake tubes at 37°C at 150 rpm for 4 hours.

8. Prepare 10-fold serial dilutions of the cells with LB Medium. Plate 100 μL of each dilution on an LB agar plate containing (50 μg/mL kanamycin, 7 μg/mL gentamycin, 10 μg/mL tetracycline, 100 μg/mL X-gel, 40 μg/mL IPTG)

9. Incubate plates for 48 hours at 37°C.

10. Pick white colonies for analysis.

11. Analyzing recombinant bacmid DNA by colony PCR.

As you can see, the result of DNA electrophoresis I can't detect the bacmid.

The primer I used tends to self-anneal.

Primer sequence:

pUC/M13 Forward

5′-CCCAGTCACGACGTTGTAAAACG-3′ pUC/M13

pUC/M13 Reverse

5′-AGCGGATAACAATTTCACACAGG-3′

I made tranformation from obligate anaerobic clostridium but it always fail. I use 20 µL of competent cell and 2 of µL plasmid (concentration is 128.3 ng/µL) with electropration protocol as: V = 700 volts ; R= 200 ohm ; C = 25 µF ; Cuvette = 0.1 cm

1. The bacteria is not growing in the plate.

2. Sometimes the bacteria growing but the gene could not inserted only antibiotic resistance band appear.

Hello everyone!

I'm trying to knock out a non coding sRNA gene from E. coli genome by homologus recombination based method. I'm using Kan cassette and pSIM6 recombination system. I grow the pSIM6 strain at 30 degree till 0.5-0.6 OD and then transferred to 42 degree water bath with continuous shaking for 15mins. After giving induction, I transfer the cells in slurry ice with continuous swirling for 10 mins. Then the electro competent cells are made by giving two consecutive washing with nuclease free water. The generated DNA cassette is electroporated by using 0.2 cm cuvettes with 2.5kV voltage for 5.5ms. After the transformation, I grow the cells at 30 degree. I get 5-6 colonies. However, after doing colony PCR with the primers for the sRNA gene, I always get band in its proper size, which implies that the mutation did not occur. I have done several times, still not getting positive result. Please suggest how to troubleshoot.

Dear colleagues,

I have a small question about competent cells protocol

Could I use the protocol to make competent cells

E. coli for S. aureus ?

If you have further suggestions or protocols

(the one that you already did well)

please share with me

Thank you so much ,

How to prepare electrocompetent cell from Staphylococcus aureus XN108?

Pgex-4t1

I'd like to know What is the minimum and maximum amount of Plasmid DNA that can be used for transformation of Bacteria ?

I gave overnight culture from my DH5alpha glycerol stock in 5 ml LB and 5 ml LB+Kanamycin as control.There was no growth in the antibiotic added LB the next day(validating that my stock isn't contaminated with a antibiotic resistant plasmid).The next day I gave 1% subculture from the 5 ml LB culture to 100 ml media and prepared competent cells using the traditional CaCl2 MgCl2 method and also performed transformation with a kanamycin resistant plasmid to test their efficiency.The problematic thing is that I got four positive colonies in my control plate(Kanamycin agar plate where just the DH5alpha competent cells without any plasmid) was plated.It is very difficult for me to trace the contamination because I did each of the step in laminar hood and also near flame and I had autoclaved everything from tips,glycerol,media,CaCl2,MgCl2 before using.Can anyone tell what might be the possible source of contamination in this case?

I have DH10b, which I have supplied as a ready-made chemical competent. Can I make this bacteria electrocompetent after culturing it on LB agar (without antibiotic)? Or do I need to make it chemically competent again?

transformation after ligation is nessesary step to get desired product in large amount.

I am facing problems with NEB Stable Cells. The original stock has an expiry date of 2020 but we thought the glycerol stocks would be fine. We are not able to clone and transform in NEB competent cells. For control we also cloned in DH5-alpha, and we were able to clone in that.

Specifically, our NEB Stable cells have lost their re-growth potential. They form few colonies in the first plate but when confirmed for positive clones and streaked on second plate they show no growth. Can somebody help?

I have been trying to amplify NEB 5-alpha F'Iq Competent E. coli for my phage display library and therefore need a high transformation efficiency. The commercial product has a TE of 10^9, but I have tried the chemical transformation cells amplification protocol (using Inoue method) and also electrocompetent method (using ice cold 10% glycerol) and got only 10^5 TE resp. Please help me troubleshoot or suggest some ideas if anybody is familiar with the strain. Thank you.

I have performed heat shock chemical transformation of pCDNA3.1 vector containing gene of interest as insert into DH5 alpha strain. Transformant colonies were observed to have grown up in Ampicillin containing LB plate.

The size of the plasmid of interest is 6.7kb, but on running gel after extraction, i always observed a band of more than 10kp using undigested sample while the R.E digested sample is always showed blank (not visible).

Please, I need an expert opinion, could it be a compatibility issue between the pCDNA3.1 and DH5 alpha cells?

I am carrying out a CRISPR knockout using PX458 plasmid with the following information

GROWTH IN BACTERIA

My question: After cloning in the sgRNA into this plasmid, can I transform the plasmid using competent DH5 alpha considering the fact that the growth in bacteria is Stbl3 growth strain?

Hi everyone.

I am working on making RNA probes for in situ hybridization, and am at the moment about to transform some competent cells. However, I just realized that the concentration of the vector that I am using (Zero-Blunt TOPO vector) is only 10 ng/ul, where I can use up to 1000 ng for the ligation in the protocol that I am following. I am not following the TOPO protocol because I want to do restriction digestion, where the TOPO vector protocol assumes that you are using the blunt ends. I tried doing restriction digestion on the TOPO vector to get the ends sticky and run it on gel, but I got no band up, supposedly because I only used 20 ng. The problem is that I guess I will have to use pretty big volumes to get a visible band on the gel (which I will need to cut out afterwards), but the amount that I have of the TOPO vector is not very large. Also, the TOPO vector I am using is already blunt-ended, so I was wondering if it is possible to first make it circular and than do some transformation on competent cells just to get more vector?

I am trying to prepare a construct using pCFD6 plasmid as the vector and 2 overlapping sequences as the insert. As per the protocol, I performed the Gibson assembly reaction in a thermocycler. Is this essential to purify this assembled product and then transform it into the competent cell?

Hello

I have used C41(de3) and transformed my plasmid. The colonies were stored in 4°C. To use the refrigerated cells for induction, am I to modify my method or follow the same protocol with 37° incubation for 1 hour, then 30 minutes cooling in 15°C, IPTG addition and overnight incubation.

Please if you could help

Thank you

I want to make my own BL21 CodonPlus (DE3)-RIPL chemically competent cells, but I don't know whether the protocol is the same as the other strains (BL21 or BL21 (DE3)). Who can provide a detailed protocol for me?

I am using INVSC1, which is S. cerevisiae with the genotype....

INVSc1: MATa his3D1 leu2 trp1-289 ura3-52 MAT his3D1 leu2 trp1-289 ura3-52

....I have cloned two genes of interest, one in psf-tefi-tryp1 and the second in pYES2. The expression vectors contain marker genes so that colonies grown in media lacking tryptophan and uracil should contain both.

I currently have a INVSc1 cell line that contains psf-tefi-tryp1 and I am wondering if I can proceed to make competent cells from here. Or should I start with an untransformed cells, transform using both plasmids, and plate on (-tryptophan , -uracil) media?

I've read that plasmid compatibility should not be an issue in yeast, but should I be aware of anything in particular?

Thank you!

Hi, recently I tried to express my genes (from plant) into Rosetta De3 pLysS using this protocol:

1. 1% overnight culture into a new media + antibiotic, incubated to grow until OD600= 0.5

2. Induce at 19°C, overnight with 0.1mM, 0.3mM and 0.5mM IPTG.

3. On the next day, 1ml culture was taken and spin to separate pellet and supernatant. Supernatant is discarded as protein is intracellular (discovered before during expression using different competent cells)

4. Pellet was resuspend in 1ml of lysis buffer (KH2P04, K2HPO4, NACl, KCl, glycerol, Triton X-100 and H2O, pH 7.8) by vortex.

5. Resuspend pellet was sonicated for 5 min (30s on, 30s off) then spin to separate soluble protein (in supernatant) an insoluble protein (in pellet)

5. Pellet was resuspend with 8M urea

6. SDS PAGE analysis was conducted upon addition of sample buffers

From the gel, there is no prominent band at the appropriate size for my genes at all. Why does this happen?

Thank you in advance for answering

I am having difficulty in obtaining transformants, the positive control plasmid pKLAC2(malE) was prepared for transformation and only small colonies were observed. These did not secrete malE protein. I am worried that my competent cells are bad and I am reaching out for any tips, tricks and advice that you might have for me to determine if they are good or not. I will try to summarize my concerns, observations and questions. Q1: Are transformants on YCB (yeast carbon base) + acetamide (sole nitrogen source) expected to grow similarly to K. lactis on rich media? Even when I plate a very small volume of the transformation reaction, I get >300 small (0.1 mm) colonies after 24 hours. They proceed to grow slowly and I am aware that untransformed cells have weak acetamide degrading ability. I have subcultured a few select colonies and they grow fine on YCB + acetamide, albeit half as well as colonies growing on rich PDA media. They must not be transformants? Q2: Untransformed cells appear to be growing better than they should be....could it be possible that impurities in agar could be supplying nitrogen for growth? Small colonies after 24 hours can't be normal but I can't determine what is weakening the selection method. Or selection by acetamide is not as selective as I thought. Q3: After adding the transformation reagent to competent K. lactis cells, they remain in small (0.1 - 0.4 mm) visible clumps and aggregate. Is this normal? The solution never quite reaches a uniform turbidity, even after the incubation, heat shock and recovery steps. Q4: Unrelated question, galactose wouldn't go bad would it? I have a bottle from at least 30 years ago. I know that glucose has an indefinite shelf life (pretty much) but I'm not certain if "bad" galactose would explain the absence of protein expression.

I just did my molecular cloning these days. But I got failed after I transformed the plasmid into competent cells and did the colony PCR and inoculation along with it. the insert is 1.5 kb and the plasmid is pET28a 5.3kb.

Actually, I have done this twice. The first time I got very blurring and light bands. I thought it may be because the TAE buffer was worn-out or the ligation buffer was not fresh anymore and the insert somehow did not get into the plasmid rightly.

However, after I used the newly bought ligation buffer, I still got the same result in colony PCR and electrophoresis.

Does anybody meet this situation and know what exactly happened?

Guys, I just got a problem with my colony PCR. The DNA template I used is ~1.3kbp and it's cDNA in a plasmid with AmpR as my supervisor said. (There is not enough of this DNA template so I want to replicate the plasmid to get more)

So I just transformed the DNA template with about ~29ng into 100 uL Top10 competent cells.

Then I spare it on an Amp+ LB plate and incubated the plate overnight.

On the second day, I got a lot of colonies. I performed a colony PCR with a positive control (from the exact same tube of the template for transformation). I picked the colony by using 10 ul pipette tips to touch and swirling a little bit and placing them into corresponding tubes. And then I pipetted the tips in the pre-mix added in the tube and swirled a bit.

But all I got is positive control and there is no band in the 18 colony sample lanes.

I am facing a MACH1 transformation related problem. My plasmid is PCMU-GAr which contains spectinomycin resistant gene. I've transformed the same plasmid in MACH1 and am getting the lawn of MACH1 after incubating the transformed cells on LB plates supplemented with 100ug/ml spectinomycin for 16 hr at 37 degrees. No plasmids were obtained later on. When I streaked MACH1 competent cell (untransformed) on LB plate supplemented with spectinomycin (100ug/ml), astonishingly the MACH1 cells showed growth. I am not sure about the fact if MACH1 is only able to do transformation when selection is ampicillin only. How to fix this problem?

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I have a plasmid with a concentration of 12858 ng/μl then I did serial 1/10 dilutions. I used my leas concentration DNA(1.28*10^-6 μg/μl). Then I took 5μl of that and added it to a tube with 25μl of competent cells. After transformation, I added 500 μl S.O.C. Total vol 530μl.

Then I did serial 1/10 dilutions to 5 tubes with a total vol of 200μl. I add 180μl S.O.C to all 5 tubes and then I add 20μl of 530μl into the first tube and then I took 20μl of that and adding to the second. I did the same until the last tub. Then I spread 50 μl of the last three dilutions of the transformation mixture onto LB plates with antibiotics. The day after I count the number of colonies. The last tub gives 300 colonies, fourth tube1071 colonies grow.

Now I should know the mass of DNA to find TE.

could you please help me? I am confused and I don't know what should I do.

Hello all,

I have been trying to transform my plasmid ~10kb in comptent E.coli DH5a cells for the past one and a half months by heat shock method but haven't been able to get a single colony. I am doing transformation by the standard protocol i.e., by thawing competent cells (50ul) on ice in microfuge tubes, adding around 5ng of plasmid, incubating on ice for 30 mins and then giving a heat shock at 42°C for 90 seconds in a water bath (also tried giving heat shock for 40 seconds) and then putting cells on ice for 5 mins again. After that I added pre-warmed 1ml LB and kept them for 1hr in shaking incubator. Then spreading it on amp+ plates with a conc. of 100ug/ml.

I can't understand what am I doing wrong. I always prepare fresh ampicillin stock for my plates and made competents cells many times by CaCl2 method but never have been able to get colonies. Can someone guide me regarding this?

Ok, so long story short. I am having trouble cloning shRNA oligos into the pLKO.3G vector. I am doing a sequential digest with EcoRI-HF and PacI and then column purifying the vector. Analysis on a 1% gel shows the vector is linearizing, therefore I'm not sure why my sequencing readouts are bad.

Annealing protocol is as follows:

1. Incubate at 37C for 30 minutes

2. Incubate at 95C for 5 minutes using heat block 

3. After 5 minutes, remove from heat source, let cool at RT on bench top. This is now your annealed oligos 

4. Make two dilutions of annealed oligos 1:10 and 1:100 for each shRNA construct 

Ligation reactions are incubated at RT for 10 minutes, followed by 37C for 10 minutes, and 65C for 10 minutes.

I then take 4 uL of my ligation reactions into 30uL Stbl3 competent cells ---> Heat shock protocol--> add ligation + stbl3 mix on Amp LB plate and left to incubate overnight.

I am afraid that maybe my vector is self-ligating which is why my sequencing read-outs are bad. I am adding SAP to my sample after digesting with PacI. I let the sample incubate for another 30 min at 37C water bath, followed by 5 minutes at 65C.

Recently I am trying to ligate a small fragment (〜300bp) to a 7K vector, but failed to get any transformants on plate.

I have checked competent cells by transforming control plasmids, DNA size by sequencing and size-checked on agarose gel after digestion. I also checked the ligation kit by doing positive control. However nothing went wrong.

Could it be possible that the issue lies on ligation condition ?(I did 4dg.overnight)

Perhaps low temperature is difficult for such bp difference of insert and vector??

I prepared a glucose solution (4 g glucose in 20 ml water) for making bacteria competent cells and kept it in fridge but it is solidified. I have never faced such a situation. Is it a normal phenomena?

Hi everyone,

I have to do qPCR to quantify microorganisms with the nirS gene but I have a problem with the standard. Indeed, due to a material problem I lost all my competent cells containing the plasmid with my standard nirS gene.

I was wondering what was the best solution in the emergency: Use an externe enterprise to synthesize an oligo corresponding to the sequence of the gene, order the reference bacterium for this gene and do the extraction and recovery of the gene...? Thank you for your help