Plates were incubated for 12 hours.
Kit info: https://tools.thermofisher.com/content/sfs/manuals/topota_man.pdf
Hi Eslam,
There could be a lot of problems. Did you have used the right polymerase to get T-Overhang? Did you use a proofreading polymerase? Did you had enough ng of your PCR Fragment? Have you purified your fragment? At this step we had sometimes problem, so we changed the kit. Was the media for the bacteria okay? Sometimes you have to add NaCl to the LB media. Which concentration of ampicillin did you used. We were sucessful with 80µg/ml.
Hope it helps.
greetings
Hi Udra,
Thanks for your concern.
I tried using the same set before with a different cell line with approximately same concentration of PCR Fragment and it worked fine.
The problem here I did not get any colonies even in the vector control plate.
I added SOC media, or what do you mean by media for bacteria?
The ampicillin I used was 20 µg/ml.
Hi Eslam,
What do you mean with a different cell line - you mean another bacteria strain? The ampicilin concentration appears quite low. In the protocoll they recommendend 50µg/ml. With media I mean the Luria Bertani media which they recommendend for the plating.. Or are the bacteria not enough competent, did you used Commercial one or did you make them competent by yourself?
Hi Urda,
Sorry for being not clear. What I meant I tried the cloning of a different cell line "PCR fragment" using same kind of bacteria "E Coli" from "TOPO 10 one shot chemically competent cells from life technologies.
I did a troubleshoot to test the quality of the plate itself by inoculation a colony from a week old plate I hope it is a matter of Ampicillin concentrations.
Thank you very much
Hi Eslam, Urda is right - 50 ug/mL amp is standard. I use 100 ug/mL so the plates are usable for longer. The higher level is fine for any pUC origin of replication-based vector. The older low copy pBR & pET origin of replication vectors are better with 50. The vector you are using is fine with either. The low concentrations of amp that you are using (20 ug/mL) will cause high background (colonies growing even without a resistance plasmid). As Urda said, most proofreading polymerases don't make the required overhang. You are better off with a non-TA TOPO kit. 12 hour incubation can be too short, try 18 hours. What exactly do you mean by vector control plate - Transformation of your competent cells with a plasmid? No colonies in that case, maybe your comp cells haven't been stored properly. Try making fresh comp cells with CCMB80 buffer.
Hi Craig,
I already changed amp Conc into 100 ug/ml and hopefully it worked.
I repeated the experiments using two different sets of ligation reactions:
In the first set, I used 2 ul of PCR fragment without any dilutions.
In the second one, I did 1:1 dilution and it gave me very good number of colonies.
Thanks Criag and Urda for your valuable help.