Can you help me for Adenovirus CPE?

I am currently investigating the CRISPR 9 case to target my gene of interest for knockout. The vector being used is lentiCRISPR v2puro. I digested and dephosphorylated 5ug of the lentiviral CRISPR plasmid with BsmBI,Subsequently, I ran a gel electrophoresis as shown in the attached image.

Following this, I excised the larger band and stored it at -20°C overnight. The next step involved purifying the gel using a Promega kit. During the purification process, I added 500ul of membrane binding solution, briefly vortexed the mixture, and then incubated it on a heating block at 88°C for approximately 25 minutes until the gel slice completely dissolved.

The DNA concentration for 5ug was measured at 30.144 ng/μl, with a 260/280 ratio of 1.70 and a 260/230 ratio of 1.26.

Next, I performed phosphorylation and annealing of each pair of oligos, which were then diluted to a 1:200 ratio. The two target sequences are as follows:

I have heard that it is recommended to add "g" to target sequences even if they already start with "g", as omitting it could result in a lack of colonies after transformation. is that correct ?

The third step involved ligating the sequences using Quick Ligase. In the ligation reaction, a 1:3 ratio was used, so 2.2 ul of vector and 6.8 ul of insert DNA were added, along with 10ul of ligation reaction buffer and 1ul of Quick Ligase, making the total reaction volume 20ul. The mixture was then incubated at room temperature for 1 hour before adding 50 ul of DH5a cells. Heat shock was performed by incubating on ice for 30 minutes, followed by a 30-second incubation in a 42°C water bath and then back on ice for 2 minutes. Subsequently, 250 ul of prewarmed LB media was added, and the mixture was incubated on a shaker at 37°C for 1 hour. The resulting solution was then spread onto LB agar plates containing Ampicillin (100 μg/mL) and incubated in an incubator at 37°C overnight. Colonies were obtained for the positive control using pUC19, but not for the sample.

During this this procedure If anyone has any insights into why colonies were not obtained your input would be greatly appreciated.

I had done calculating MTT assay using

Among them which one is the best and suitable for calculating IC50 value. When using grpahpad prism different parameters were available, which is applicable?

Thanks in advance.

Hello, everyone. While transfecting my cells with plasmid, I noticed this overnight; normally, my cells are adherent (as seen in the background); I'd like to know if anyone else has seen this before; is it a contamination? I didn't see any media color changes. and this is a floating separate component, not just one. Thank you for any suggestions.

Hello everyone,

I have a question about the MTT assay. In the attached image, why do some cells show MTT color while others don't? Some wells only display color around the edges. All cells appear intact under the microscope.

Has anyone encountered this issue and found a solution?

Thank you for your assistance.

Cell used-C20 cells

Treatment time- 48 hours

Control Seems ok, for other well I have used different concentration of Curcumin. (100uM to 0.01 uM)

Protocol-

MTT assay

Hi All,

I have noticed a consistent trend of low optical density readings, ranging from 0.4 to 0.6, for the negative control MRC-5 cells in our experiments. This observation has prompted me to inquire about the typical range of optical density values obtained for negative control MRC-5 cells in other studies.

Our MRC-5 cells demonstrate robust growth characteristics, having been sourced from a recent batch obtained from our supplier and cultured in accordance with the recommended EMEM medium protocol provided by ATCC.

I would greatly appreciate any information or experiences that other researchers can share regarding this issue. Thank you

Dear ResearchGate community, hello!

I'm going to do an MTT test on glioblastoma cells with our drugs. I plan to add 10,000 cells in 100 µl medium with FBS per well to a 96-well plate, incubate for 24 hours and then add 50 µl of drugs. Incubate the cells with treatment for 24 hours and add MTT dye.

Tell me, please, should I remove the medium before adding the drugs (they are diluted in DMEM without FBS) and should I remove the drugs before adding MTT dye?

Thank you in advance!