Transformation (Genetics) - Science topic

Hello

I am currently engaged in research focusing on plant genetic transformation. As part of this endeavor, I have designed a comprehensive in silico plasmid cloning approach. The aim is to enhance the accuracy and efficiency of our genetic engineering efforts in plants.

I welcome and deeply value input and suggestions from all stakeholders involved in this research. Your insights and expertise are crucial as we strive to optimize our methodologies and achieve impactful results in the field of plant genetic transformation.

Together, through collaboration and exchange of ideas, we can ensure the effectiveness and precision of our approaches, ultimately advancing our understanding and application of plant genetic engineering for various beneficial purposes.

Anyone is invited to write me at shupty2010@gmail.com

Thanks in advance everybody

I am co-transfecting NIH3T3 cells with two plasmids (rasV12 mutant + gene of interest) for a transformation assay. My question is: how much reagent (FuGENE HD) should I use? I typically use a 3:1 reagent:DNA ratio for single transfections. But as I am adding twice the amount of DNA in total, should I use a 3:1 ratio for only one plasmid or both plasmids? Out of situations A and B below, which would you recommend? 

Situation A: rasV12 (1 ug) + Gene X (1 ug) = 3 ul FuGENE HD

Situation B: rasV12 (1 ug) + Gene X (1 ug) = 6 ul FuGENE HD

Dear all,

I need a stable expression of the OVA gene in B16F10 cells. I've tried introducing the OVA gene via retrovirus transduction, but it doesn't seem to be working. Transduction on control L1.2 cells worked fine, suggesting that there was no issue with the viral titer and transduction protocol. 

Has anyone successfully done a retrovirus transduction on B16F10 cells and would like to share the protocol with me? Would it be easier for me to just move on to try lentivirus?

Thanks! Any suggestion will be greatly appreciated!

Regards,

HS

I am trying to genetically transform a species of Aneurinibacillus. I have tried protoplast transformation, electroporation, and natural transformation, but have not been successful. Does anyone have a good protocol for this? Thank you.

I wanna ask about procedure in genetic transformation procedure. I am conducting the transformation using A. tumefaciens but have difficulties to control over growth of bacteria. I am usually using OD600 0.3-0.4. In the first and second selection medium the bacteria is good, no over growth. When I transferred it to third selection medium the problem appears. Most of explants affected by A. tumefaciens. How should I do with my procedure?

Thank you for your responses.

I wonder if exists a protocol, or any project, focused on bioplastics production using avocado, reaching that goal by a genetic transformation with plasmids (binary or not), which might include genes involved in bioplastics synthesis (for example, including genes for polylactic acid-PLA production). Thanks.

I want to do plant genetic transformation with my gene of interest. For that reason, I need to construct the vector Ti plasmid so that can go for Agrobacterium-mediated plant genetic transformation. How I will cut the plasmid, what will be the enzyme and how to make it ligate? Anybody who has already done such work can help me by providing me the protocol?

Thank you in advance.

Can anyone suggest me how to avoid fungal infection on regeneration media while doing agrobacterium mediated transformation of arabidopsis leaves by co culture method?

For agro removal i prefer to use cefotaxime and that works well but i immediately get fungal contamination hence no further callus initiation. This has happened several times with me in case of tobacco and arabidopsis both.

Hi, 

I'm trying to disrupt some genes of the genome of E. coli. For achieving this, I'm following the protocol of the P1 phage in which this phage transfers genetic material from one strain to another. The recipient strain is from the keio collection, so I don't have any problem with the selection of the colonies which were succesfully trasduced.

But after the confirmation of the disruption with the antibiotic screening and PCR, I tried to remove the Kn resistance using flippase. The flippase is a recombinase that recognize FRT (Flippase recognition target) sites and removes all the flanked area.

This plasmid must be electroporated and then grow it at 30°C, because it has a temperature sensitive ori.

I have done all of this but I have not obtained any transformant. Do you have any clue of why I don't get colonies? Can you give me some tips?

I am trying to assemble multiple constructs:

- 2 fragments (1300bp and 2000bp) into a 3.3 kb vector

- 1 fragment (2000bp) into 5 kb vector

- another 2000bp fragment into the same vector

- 1 fragment (3600bp) into a different 5 kb vector

All of them are failing, as in: giving no or very little colonies. I've wasted quite a bit of this expensive master mix so far, and am running out of options as to what to try. Sometimes I get 1 or 2 colonies, but never the expected amount and never with the correct insert.

The most logical thing based on these observations is the competent cells being bad, but I've transformed them with an intact plasmid and got 200-300 colonies on the first try, so they are obviously fine.

The next sensible thing would be that my ligation is simply very difficult for some reason, but in that case, it wouldn't be failing for all 4 constructs.

I linearise my vectors by PCR and gel-purify them. I have tried with purified and unpurified PCR products.

I tried assembling the positive control in the HiFi mix and got 3-5 colonies, which is still way too little (manual says >100). So I used another vial of HiFi mix, thinking maybe the enzymes were bad, but I got exactly the same result.

I've used this same method multiple times in the past to assemble constructs of higher complexity than this, and it has always worked in the past for me... What am I missing here? Should I sacrifice a loved one?

Thanks for your time!

I have transformed rice plant by Agrobacterium mediated gene transfer system but getting problem of contamination by fungus. Is there any problem if I use antifungal in that plate to get rid of fungus. If so, what commercially available anti fungus I should use. For more, my construction doesn't have any gene that can make resistance against antifungal.

Hello there !

Currently I'm running out of ideas of what can be improved regarding the handling of my research and now I would love to have some input from you out there !

I want to transform the Bacillus species Bacillus Firmus and after several attempts of protoplast transformation I've got access to an electroporation machine. (I cancelled protoplast transformation because most of the time they are just dying indicated by missing colonies on non-selective agar plates and I'm not able to find hints in terms of how to suspend bacterial protoplasts without handling them too harsh).

Please have a look in the files. There are two pdf files: The first one describes my very own handling of the protocol I'm working with, calculations, plasmid informations and a troubleshooting. The second file is the actual protocol I'm working with.

If there are any informations missing please let me know. Also feel free to check the calculations. I'm a bachelor student and maybe I made silly mistakes. That would be shameful but at least I'd know what went wrong.

Thanks you so much !

Daniel

I'm doing SDM on a pENTR vector (2.8 kb with my gene). I get colonies (using Gold competent cells) on a plate with kanomycin, but when I grow one of these colonies in broth I can't isolate any plasmid. I've also done negative controls: un-transformed cells without the pENTR vector can't grow on the plates.

The cells are definitely growing in culture, but when I check on a Nanodrop or on a gel, no DNA is present. 

I made a new stock of kanomycin (50 mg/ml) and filtered it through a .45 um filter. Our powder of kanomycin sulfate isn't expired. I've also tried using a higher concentration of kanomycin (3X) but that hasn't worked either.

Any ideas as to why this may be happening?

I m using dh5 alpha cells to insert some specific segments from influenza A . I ligated the cells using TA cloning kit and transform them . I cultivated the cells on an LB media with ampicilin , Xgal and IPTG for the blue white screening. The screening worked perfectly , I cultivated them overnight and after this I used the Qiagen mini prep kit. I used ecor1 to digest the mini prep and after this i run a gel electrophoresis (agrose at 1%) for 45 minutes at 70V . I can't understand why I got the top bands like this , is it super coiled DNA ? Is something normal or I didn't do any of the steps properly . Does somebody know where I failed ?

There are many ideas to create genetically modified microalgae for higher productivity,

Why are the reports on this so little?

I think now it is more possible with CRISPR technology.

Any idea of what are the barriers?

Hello :). I have to put my plasmids (dh5alpha) in LB medium containing spectinomycin. Whar i´m wondering is which is the right antibiotic´s concentration due to the fact that my plasmid is a low copy number one?

I checked some articles and they have used an spectinomycin concentration of 100 ug/ml but i don´t know if it is too much for a low copy number plasmid as mine.

Thank you!

Hello,

Can anyone please provide me a detail protocol for coating gRNA and Cas9 protein onto gold/tungsten particles for particle bombardment in plants.

Thanks!

My goal is to insert 16 kb of plasmid DNA into S. cerevisiae by transformation using electrophoresis but the result showed no colony on selected plate. Could anyone suggest me about my protocol?

>Here is my rough protocol

Competent cell preparation

-yeast OD. 0.7-1.2 > centrifuge and discard the supernate (4c) > wash with ice cold water 2 times > treat with 0.1M LiAc + 10xTE + 1M sorbitol at 30c, 30 min > treat with 1M DTT at 30c, 15 min > wash with ice cold sorbitol 2 time >re-suspense the pellet with ice cold sorbitol + 15% glycerol and aliquot 40 microlith per tube and keep at -80c until used.

Electrophoresis

-Thaw 40 microlith of yeast cell from -80c on ice 5 mins > add 200 ng of plasmid DNA and transfer all solution into ice cold 0.2 cm-gap electroporation cuvette > pulse 1.5 kv, 25 microfarad, 200 ohm (when I finished pulsing, time constant report showed approximately 4.2) > add 1 M ice-cold sorbitol in to cuvette and move all solution into tube 1.5 ml sterile > recovery yeast at 230 rpm 30c, 1.5 hour > spread on selection plate and incubate plate at 30c (observe colonies everyday until 5 days)

-I used media which dropout leucine for a selection plate. It contains Yeast synthetic drop out medium supplement (lack of leucine), yeast nitrogen base without amino acid and ammonium sulfate, agar, water and sterile glucose was added after autoclave.

(Is there any differences between Yeast nitrogen base without amino acid ammonium sulfate VS Yeast nitrogen base without amino acid but contain ammonium sulfate?)

Thank you =)

What is the most efficient protocol for transforming bacterial cell, say BL21DE3 with plasmids carrying protein construct?

Anyone know of the impact of timentin on in vitro cotton regeneration? I did not find enough material on the subject.

Currently I use cefotaxime to control Agrobacterium after genetic transformation and I imagine that Timentin has a similar effect, perhaps with a greater control over the bacteria.

I wonder if the effect on cotton tissue would also be similar to that of cefotaxime or whether there is any negative effect.

Hi everyone!

I have a problem with e.coli cultivation.

I use pET28a and pBHA plasmids. My gene is in pBHA plasmid

pET28a consists of NdeI and XhoI restriction sites.

An insert also has the same restriction sites.

After restriction electrophoresis shows restriction products:

Gene approx - 400-450 bp ( the gene mass - 420 bp)

Restriction product from pET28a - 80 bp (as it is described in standard manual)

After ligation of a gene and an uncircular pET28a the electrophoresis shows products of ligation that look like products of polymerisation of (1) insert (Gene =420 bp, but the major ligation product - 2000 bp by using MassRules DNA Ladder Mix as a marker) and (2) the vector (near start position).

No product in an expected area of 5000-6000 bp persists.

No colonies after 16-20 hours of incubation. Only the positive control (non-restricted circular pET28a) gives many colonies.

I've found that the gene consists of 5-CATATG-3 fragment - classic NdeI restriction site, and 5-GTATAC-3 - the opposite fragment.

The problem is that:

The process of restriction persists (you may see a gene next to 400-450 bp, and previous pBHA vector aprox 2000 bp)

When I use a non-restricted circular(!!!) pET28a it grows! When I put it in a ligase buffer and do not inactivate ligase it also grows!

Negative control (restricted pET28a) is negative.

Thats why I have some questions:

Where can be the problem?

Is it possible, that NdeI that cuts fragments 5-CA'TATG-3 may do it in case of 5-GTAT'AC-3?

It may explain why vector do not react with insert in case of another unusual NdeI site

I am looking high efficient genetic transformation method and direct shoot regeneration through explant without investing more weeks. So that I can reduce my experiment time.

Could anyone share the knowledge about potato genetic transformation method proving high regeneration efficiency?

Thanks

Hi,

I am using C3H10 and MC3T3 cells both from mouse origin. Im using trans gene 9 transfection reagent for transfection. But the efficiency is very low. I am using 600ng DNA / well for 24 well plate. Can someone suggest any modifications?

Thanks,

Jaya

I'm interested in trying the protocol from Sugita et al 2014 to study gene expression in the organelle. They are the only lab that has published results (a single paper) with the protocol so I'm hesitant about it.

I am trying to purify a certain protien

i transformed into BL21 competent cells and do the purification steps but after the Ni affinity column and trying to run on ion exchange there was no peaks and SDS page was done but showed no bands

knowing that i did colony pcr to make sure that the gene is transformed and it was successful .. so any suggestions ?

please add links to get more information

For the research of Agrobacterium mediated genetic transformation I need to construct plasmid vector.

If I design that vector using a suitable software, after that how I could make the construct.

Or anybody can co-operate me in this aspect by providing the construct according to my need? That will be a collaborative work.

I'm using the QuickChange II SDM kit with complementary primers to make expression vectors carrying a mutation

My vector is about 9 kBp and I follow the standard SDM protocol from the provider of the QuickChange II SDM kit. After PCR and DpnI restriction I transform 2 ul of my PCR product in XL-10 gold bacteria. 

After miniprep on the picked colonies I run an agarosegel to check my DNA. And this is where I see that the majority of the DNA I preped migrates at a size different from the original template vector (used for the PCR). Since this piece of aspecific DNA is always of the same size, I suspect a systematic error. When sending this DNA for sequencing with primers that are designed to sequence the region surrounding my mutation, I get no sequence.

Does someone know how to solve this problem?

I have an inserted construct into BL21 (DE3) pLysS expression vector, which is in the frame and has the size of about 27 KDa. I followed below conditions:

1) Overnight growing of a single colony of the transformed expression vector in 1 mL LB containing 1 micro gram/ ml Kanamycin, at 37 degree in the shaker.

2) Taking 500 micro L of the overnight culture and incubate in 10 mL LB (containing 10 micro gram/ ml Kanamycin) for 3 hrs at 37 to reach the OD600= 0.550

3) Take 1 mL un-induced sample, and test different concentration of IPTG (0.1mM, 0.4mM, 1mM, and 1.5mM) at different degrees (37, 30, 16) with shaking speed of 160-200.

4) OD600 of each sample at each step was measured and reduction was observed in none of the samples (which may refers to the non-toxic state of the expressed construct for the bacteria).

5) Samples were taken until four hours for 37 degree, until six hours for 30 degree, and after 21 hours for 16 degree.

After all, un-induced and induced samples have no difference on the gel!

I am suspicious that maybe IPTG is not working well that nothing is expressed or perhaps there is a problem in the bacteria cells. Because when the single colonies of the BL21 pLysS transformants were grown onto the LB+Kan plates the colony sizes were all bigger than the single colonies of DH5a strains. (for example if DH5a is 0.1mm in diameter, BL21 pLysS colony's diameter is about 0.3mm)

In advance I appreciate your kind suggestions and comments.

If we want cell to accept vector dna in transformation, we treated with calcium chloride or chilled on ice etc.. But i have a trouble with one issue about that. Books say that ; We must choose the vector that specific according to the cell that will do transformation. But we will treat with calcium chloride and ice, why we choose specific vector?, this foreign vector can be enforced the cell for accepting. I know cell might accept the vector as a homolog sequence and might do cross with this vector, might take homolog genes into own dna, but it doesnt have to be. Vector might stay in the cytoplasm and i think it dont require the homolog sequences with cell's own DNA . Transformation should occur spontaneously due to bacteria nature, already

Can anyone share an experimental procedure for the direct transformation of a bacillus bacterium using PCR product or other non-circular DNA fragment?

I transformed DH5 alpha with YCplac22, but I am not able to get the actual size on gel. Should I digest it first?

I am planning on transforming agrobacterium (strain GV3101) via electroporation with the plasmid pBINPLUS containing a 13 Kb insert. The insert also contains some repetitive sequences (that could not be avoided) so I am especially concerned about validating that the correct plasmid is in the agrobacterium and hasn't undergone recombination. I am looking for recommendations of how to screen the colonies - from what I understand, it is not easy to do colony PCR or minipreps on agrobacterium, and especially as my sequence is so long PCR would not necessarily be informative. 

Thanks in advance for any advice.

The strain is required for monocot transformation

For the past 2 months I have been working on a double base pair substitution, for a single amino acid (AA) change; in a plasmid that has been cloned successfully into E. coli before. Initial tests showed positive results however, further screening of the mutant colonies revealed that the substitution has no occurred; through a digestion of the DNA. 

To prevent template DNA from escaping through the concentration of DNA was reduced to the 1ng/uL. After the reactions I've been checking the PCR product which is present, but in a very low concentration as one would expect with using a low 1ng/uL concentration. However, after KLD treatment and transformation I am getting zero colonies on the 100ug/mL ampicillin plates. 

My Plasmid is an 8kb plasmid and I have been using the following reaction for the PCR: 12.5uL Q5 Hot start, 1.25uL 10uM Forward Primer, 1.25uL 10uM Reverse Primer, 1uL 1ng/uL miniprepped DNA, 9.0uL MqH2O. With these settings on the Thermocycler: 98C – 30 Seconds, (98C – 10 Seconds, 62C – 30 Seconds, 72C – 4 minutes) x 25, 72C – 2 minutes, 4C – Hold.

For the KLD treatment I have been using the following reaction: 1uL PCR product, 1uL KLD enzyme, 5uL KLD enzyme mix, 3uL MqH2O. I have tried the transformation using both the NEB Competent Cells and Top10 Cells, with both a standard Heat Shock transformation protocol and the NEB heat shock transformation protocol.

I was wondering if anyone had any suggestions?

I need to design primers and gRNA for one gene to transform it in S.cerevisiae and I am confused about it.

aa

Hi everyone,

I have a question about How we can predict miRNA binding sites on lncRNAs. Some papers recommend generally some websites such as miRanda, TargetScan or PicTar, but I think none of them are not specified for lncRNAs, I have recenty read a paper named spongeScan: A web for detecting microRNA binding elements in lncRNA sequences, I found this web maybe the useful one that I am looking for. but I have some problem with using it; when I try to load my desired lncRNA FASTA file in corresponding place I always get this message'' The FASTA file you have uploaded does not have the proper extension, please check it" but I tried in many different ways and still I get this message, Has somebody worked with this web or has any experience with this? what is the problem? and also How can I use " Get from Ensemble" option? While I select this option only I have to see " Homo_sapiences GRCH 38.ncRNA, but I want to put or select my desired lncRNA not all. Could you please guide me how I can use properly this web or are there another suitable and informative webs regarding this matter?

Thanks in advance. 

Is there anyone who could provide me information concerning recent developments (protocols) in genetic transformation and regeneration of Squash (Cucurbita peop ..)

Hello,

I'm transforming two plasmids (dominant negative Gai2 and Gai3) from cDNA Resource Center. The protocol that came with the plasmids suggests using Top10F' cells from Invitrogen. At the moment, our lab only has Turbo Competent cells from NEB, so I used them and the protocol from NEB to do the transformation.

I got a lot of growth for dn Gai2, but for dn Gai3 I keep getting only one colony of cells. Do you think it's because I used Turbo instead of Top10F' cells or because something is wrong with the dn Gai3 plasmids since the dn Gai2 grew just fine? 

Thank you! 

Is ''gene construct is a segment of DNA (T-DNA ) to be introduced in a target organism'' or does it ''includes the transformation vector DNA also i.e., 'segment of DNA to be transfer + the remaining vector sequence'.''?

What in case of a Double T-DNA harboring transformation vector (a vector containing two separate segments of DNA to transfer), will it be called to have two gene construct or two gene cassettes?

I am trying to overexpress proteins in Bt. I would like to transform Bt by electroporation. I have protocols to do that but I am not sure about the plasmid characteristics

Hello, everyone, I want to create 6 SDM at a different location in the coding region using 6Kb Plasmid. I had chosen 3 different sites among 6 sites and started to mutate using QuikChange II XL Site-Directed Mutagenesis Kit.  

I used below mention PCR (18 cycles) program I got the colonies.But unfortunately, all colonies are wild type even I changed the site of mutations but still, all are wild type.

94 for 3'

94 for 1'

52 for 1'

68 for 6'

68 for 60'

4 for hold  

Step2---Dpn-1 digestion at 37 degrees for 1hr.

Step3--- Purification of PCR product using the phenol-chloroform method.

Step4 -Transform all volume approx's (8-10ul) in 50ul DH5alpha.

Step5-Saturate the transformant for 1hr at 37 degrees.

Step6-- Plate on LBA with Amp+ incubate O/N. 

Step7-- Inoculation in LB with Apm+ for O/N. 

After sequencing, all are wild type.

Could any one share an idea to do 6 SDM one after other?

I am trying to isolate a regularly sized plasmid from a standard DH5 alpha strain. However, I keep yielding very low plasmid copies.

I am pre growing (liquid media) the bacteria in penicillin before the extraction as selection marker. However, I am wondering whether the antibiotic could be effecting the yield of the extraction in some way ... does anyone know if this is the case?

For reference, I am using a standard promega wizard plasmid extraction kit.

I am working with the E. coli strain BAS901, which has a mutation in lptD rendering the cells hyperpermeable via defective LPS assembly. I need to introduce a plasmid into this strain. Unfortunately, the standard protocol I use to make electrocompetent E. coli cells is not working. I've even tried preparing DH5alpha electrocomp cells at the exact same time, using the same procedure. The DH5alpha cells came out quite competent, and the BAS901 strain is totally unable to take up the same plasmid.

My procedure for making electrocompetent cells basically involves growing cells near and OD600 of 0.4, spinning down, and washing the pellet several times with distilled water. Finally I resuspend in a small amount of water with 10% glycerol. The entire procedure is done on ice.

Is it possible that, because BAS901 is a hyperpermeable strain to begin with, I'll need a different procedure for creating competent cells or performing the electroporation itself?

The successful binary vector is P. Cambia 1301 and is very efficient. All are around the same size (11kb), contain hygromyocin and streptomyocin resistance, and have been confirmed by restriction digestion from E. coli midiprep. After a third try with the 2 failed b.v.s I had one colony grow on the Ubiquitin B.V. plate... but was told that it could be a fluke.

Any advice on why P. Cambia works but my other two will not?

I am working on TG1 competent cell preparation. An uausal protocol was used. TG1 cell was cultured in 1L YT medium containning 20mM MgCl₂ and When OD600=0.5, the cell was transferred to a clean container and cool down on ice for 30min. Spin down the cells and wash the cell with sterile water twice,10% glycerol once. Finally resuspend the cell in 1ml 10% glycerol.

I introduce a 5000bp phagemid into TG1 competentent cell. I am using 1mm cuvette,100ng DNA in 50ul competent cell.  Ampicillin selection was applied and the transformation efficiency was 5*10E8 per ug DNA. It is a little bit lower if I use it to construct a library. Do you have any suggestion to improve the efficiency of competent cell? Thanks.

I am trying to generate some mutant clones via MARCM. Not successful yet, a bit worried if my stock has lost its FRT site while still retaining the mutant because they have not lost the balancers. The mutant is homozygous lethal.  

Different methods are available for removal of selectable marker gene after transformation in the case of rice like co-transformation through the 2-vector system, super-binary vector system etc.

I am working on a flagellar gene mutant of chlamydomonas which gives palmelloid like growth in liquid media. Is it necessary to hatch them to single cells or can i directly take the clumped cells for electroporation?

Ecological risk of biofertilizers

Can anybody recommend a good commercial binary vector that can be used for Arabidopsis transformation? I currently use some modified unsequenced plasmid and I would love to change it to something that actually works. 

Thank you in advance

Alin

I'm trying to generate an adenovirus of the construct of my choice by working with the pAdEasy-System. First steps worked well, I got colonies after transfecting it into the BJ cell line and the restriction with PacI showed a ~30kb band and a 3 kb band (or 4.5 kb). I took 1 µl, 3 µl and 5 µl of the miniprep and transfected it into Top10 electrocompetent bacteria but got not a single colonie. I've already tested the bacteria and, as you see, tested several amounts of DNA. Does someone have an idea what I can change or do better to get colonies? 

May be a general question; sometimes stupidity too....

For example, cancer is caused by somatic mutation in the genome. And treatment has been given to the patient. So what is the chance of change in the somatic mutation if cancer got cured?

Some one please help, I have amplified a 2.3 kb fragment (contains TAP and Kanmx cassete) pYM13 was used as template with overlapping primers. I followed this paper (https://www.ncbi.nlm.nih.gov/pubmed/15334558) for the experiment.

After transformation Can i do selection on G418 plates?

Do i need to transform in haploid cells or diploid cells? and Why?

The application is educational, for a cloning/genetic engineering project for 2nd year undergraduates. I ligated a GFP insert into a pETBlue-2 backbone and transformed into NEB5-alpha - this worked totally fine with blue/white screening and everything. I miniprepped positive colonies (white, and with colony PCR), sequenced to confirm, and then tried transforming 25ng into BL21(DE3) and got no colonies with 10^-1, 10^-2 and 10^-3 dilutions on LB + 100ug/mL Amp. I have now plated 200uL of undiluted cells onto LB + 50ug/mL Amp and just LB. Any recommendations? We are wanting to run the whole project starting in September but can't do that if we can't find an expression line that works. Cost is an issue, to a point. Thanks!

Hi everypne

Now I am using viral-TMV vector to infect Nicotiana benthamiana rdr6i mutant using agroinfiltration transient expression.

The viral-TMV vector that I used has RdRp component.

Now I will do a presentation, but I donot know why I use Nicotiana benthamiana rdr6i mutant to produce my protein, why donot use Nicotiana benthamiana WT.

If somebody happen to know, can you help me?

Thank you in advance

xiao

Why use special competent E.coli for gateway plasmid clone? What's the difference between the gateway competent E.coli with normal competent E.coli? 

I am facing a very unusual situation with my project. I have transformed microalgae with pCAMBIA1303 containing a gene construct through Agrobacterium-mediated gene transformation. I did molecular analysis of the positively selected microalgae with hptII and gus primers which showed positive results but when I checked with gene specific primers I got no bands. I am pretty sure with my PCR reactions as I got bands in positive control. one of my senior who was working on the same gene, faced the same problem, but the unusual thing he found was that when he did expression analysis he got positive results. The length of my gene of interest is 2.14kb. I have no clue for this unusual situtation where genomic DNA shows no presence of the gene but cDNA pool shows the presence of expressed mRNA. please help me.

I wonder the efficiency of Cre-loxp recombination to recombine 3 kinds of plasmids. It seems that in Multibac system, 3 different plasmids can recombine, and the positive recombinant can be selected by antibiotics screening. Who has such experience? Would you please share it? Thank you a lot.

I am trying transformation of Bacillus aryabhattai using commercial plasmid pHY300PLK and have tried lot but not getting any positive transformants, kindly help if any one has got expertise?

Transformation was done at 2000k and used TC - 4.5 to 5 

Normally, after plant transformation (e.g. callus); 50% of the infected calli undergo GUS assay, while the remaining 50% are co-cultivated / cultured for regeneration.

So, What is the method to determine the transformation efficiency percentage of the remaining 50% calli that were co-cultivated or transferred to regeneration media ?

I have just purchased a 12 rxn C43 competent cells kit.

I want to make a stock from it and store in -80 .

After i regenerate the cells by plating them out and culturing in liquid medium, to restore competency can i follow any protocol of E.coli competency and has anybody come across a study comparing protocols, because while similar they differ in small details , or from your own experience and concluded on a mot efficient protocol? 

Much apreciated,

Cheers

Hi,

I would like to measure the transformation frequencies of antibiotic resistance genes in streptococcus pneumoniae without inducing competence. Previous studies have used Competence stimulating peptide (CSP 1) for inducing competence. Is that an absolutely necessary step? Or is it done just to increase the transformation frequencies to experimentally observable values? I will be very much thankful if any reference links are provided. Thank you.

I'm trying to transform E.coli DH5 alpha by pEKEx3 plasmid. I want to know if I can use spectinomycin dihydrochloride pentahydrate instead of spectinomycin. of course I have considered the difference in working concentration.

heat shock transformation didn't work. Does anyone have experience using this plasmid?

I would be greatly appreciated if anyone could help me on this matter.

I need a reference or protocol that works well today. 

what is the difference between ecoli plasmid and agrobacterium plasmid

Hi, recently I am doing the recombinant expression by using pET-44a expression vector. After cloning step, the insert was confirmed inserted. The recombinant plasmid was then transformed into Rosetta gami B(DE3) pLysS competent cell. The antibiotics I used on LB agar plate were: ampicillin, tetracycline, chloramphenicol, and kanamycin. After incubated in 37 degree C overnight, no colonies can be found. Anybody can tell me why? Thanks a lot.

I tried to transform DH5-alpha chemically competent cells by pCMV-dest BacMam vector from ThermoScientific (10 Kb) for storage (without insert or entry vector) on LB Amp plates. It gave no colonies. I used 500 ng/ul vector on 100 ul competent cells and spread it on 2 plates (50 ul and 400 ul). 

I am working with pathogenic E. coli (not ATCC/lab isolate) and would like to create mutation. I transformed pKD46 using calcium chloride - heat shock and electroporation methods. Plates were incubated at 30 degrees. But i couldnt get the transformed colonies. Please send the detailed protocol for transformation of pKD46 in to E. coli. TIA

Hello! Could anyone help me figure out how much (ug) Cas9 protein and sgRNA should I use for each round of bombardment in plants? I need to transfect somatic embryos by biobalistic transformation.

I am a PhD student at the beggining of his research. I want to use the pCR8 / GW / TOPO vector to clone, but I have a doubt about the resistance gene of this vector. According to Thermofisher genome map it confers spectinomycin resistance (Web nº1) but, I have found that this vector confers resistance to spectinomycin and streptomycin (Web nº2). Could this vector confer resistance to streptomycin? Has anyone used this vector with streptomycin? Thank you in advance for your support.

Hello everyone,

I have to do tobacco transformation using pGreenII 0229 binary vector system. Does it works the same as that of any other binary vector in tobacco? can anyone provide me any advice or suggestion regarding this? Is there anything I should now about before starting the experiment? Can anybody provide me an reference article about it? 

Hi

I have experiment with Klebsiella oxytoca for gene integration by homologous recombination.

I have designed the linear DNA cassette with target gene and Cm. as selection marker. And The homology sequences were 300bp in front and 60 bp at other side.

The transformation was done by electroporation, and the universal integration procedure was used.

In plate incubation, I can get about 100 colonies   in plate with selection antibiotics.

However I can not get any success in gene integration at a specific site. 

(I can check it by colony pcr where the band will be detected only when the success in gene integration. but I can not any band in colony pcr)

If you had similar problem and solve it, plz help me.

Thank you.

I am trying to express sfGFP in Pichia Pastoris using the pPICZ-A vector. I have encountered challenges earlier with transforming my yeast, contamination and antibiotic selection. Here are some specifics:

Strain: GS115

Fresh electrocompetent cells cells are transformed with ~10ug linearized of my pPICZ-A/sfGFP construct and plated on YPD+2xZeocin plates. After 4-5 days growing 28-30C, replica plates are transferred to Minimal Methanol + Zeocin plates, where they are grown for a further 4-5 days with the addition of 200ul methanol to the lid of the plate, daily.

At this point I am able to take my plates to a dissecting microscope equipped with a Nightsea fluorescence adapter and see a faint phenotype in green coloration on my plates. I am then able to grow 5mL cultures of these, in YPD with the addition of 0.5% methanol for 4 days and see a consistent change in fluorescent response versus control cultures (I can provide pictures of all of these if requested).

When I take these cultures to the 100ml scale, I am unable to harvest any fluorescent protein after growing in BMMY for 5 days.

Are my green phenotypes false positives? Is the approach of screening for integration and expression using induction and fluorescence on a plate a bad idea? Notably the fluorescent response was subtle and not nearly as vivid as GFP in bacteria, but the differences were clear. I am repeating my BMMY expression with other hits from the plate with freshly prepared media. Contamination of BMGY/BMMY was a concern, and I have been since routinely adding ampicillin, which does not seem to affect Pichia growth.

agrobacterium mediated transformation

Hello, I have some inquiry. Can someone help me? Thanks for your time. Have a nice day.

Hello everyone. I have some problems. Can someone help me? Any tips please? Thanks for your time. Have a nice day.

Can i use ampicillin sodium salt instead of carbenicillin or cefotaxime for selecting transformed plant? i want to use ampicillin as it is cheaper than other antibiotics....

Thank u

Hi,

I have done yeast signal trap assay to identify secretary proteins. I have cloned my target gene (only one gene) into YST vector and transformed into invertase mutant yeast. When I culture the transformants on sucrose medium, about 50% transformants grow on sucrose medium and the rest do not grow.  How to interpret this observation? Is the gene code a secretary protein? or something wrong with the transformation? Experts, please help me…