Danny Had

10 Questions 6 Answers 0 Followers

Questions related from Danny Had

How to best design shRNA?

this is the first time I design shRNA and I have looked online but couldn't find the answer to the following questions: - should I add an overhang? what overhang do you suggest? dtdt? - I am...

24 October 2023 3,400 0 View

How to know if CRISPR did heterozygous or homozygous knock-out?

Hello everyone I have been doing CRISPR to knockout a gene from my HEK-293 cell line. I have transfect with SgRNA-cas9 and now my cells have been growing for a week. I am planning on doing DNA...

09 August 2023 4,380 12 View

Cloning sgRNA into Px458, no successful cloning so far?

I have been tring to clone a 25 bp(5`-CACCGXXXX....-3` and 5`-AAACXXXX...C-3`) sgRNA into Px458. I have been using this method :...

28 April 2023 3,610 3 View

Can I visualize a 78 bp DNA fragment on agarose gel?

Hello I am trying to cut a segment from a plasmid that is around 80 and I wonder if I could see it on the gel taking in consideration that the smallest size on the ladder is 100 bp. however, I...

26 April 2023 9,346 3 View

Is it OK to leave LB media with antibiotics in it on the self in room temprature?

I usually pre-prepare media with antibiotics and put in on the shelf for quick access (like doing minipreps). I wonder if that would effect the antibiotics in the media and if it would cause any...

12 April 2023 6,366 3 View

Can I use the same sgRNA or siRNA that was used previously in a published study?

I want to do CRISPR and ShRNA for few genes and I came a cross few studies that knocked-out those genes and they shared in the method sections the sequences that worked. usually for CRISPR and...

28 March 2023 6,101 0 View

Any advices about generating CRISPR-KO cell line?

I am trying to knock out a gene in a cell line using CRISPR and I have been reading alot about that. I want to use 2 gRNA (crRNA and tracer RNA and not sgRNA) to do a large deletion in a gene. can...

17 January 2023 5,263 1 View

What is the meaning of having a DNA with good amount that is not visible on gel?

I have a plasmid in a concentration of 50 ng\ul. I tried to run the gel and loaded 10 ul of that but nothing appears on the gel ( except for the ladder). any explanation for that?

04 January 2023 7,685 3 View

Weak e.coli lysis despite everything?

Hello everyone! I have been trying to do a Maxiprep on my e.coli but I was unable to get any plasmids and the reason is that my e.coli are not being lysed whatever I do ( ex: increase Lysis buffer...

22 December 2022 6,190 2 View

On DNA gel electrophoresis : can you depend only on the size of the plamid to determine that you have the right colony?

I did transformation for some plasmid into E.coli and spread it on agar with antibiotics, then i chose a colony and did DNA extraction (with miniprep) and then i ran the gel. for some reason i am...

02 November 2022 1,394 6 View