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.
have you heated the colony at 100oc for 5min, then spin down to take the sup into the PCR reaction?
how much did you add to the PCR reaction, and what is the total volume of your PCR?
Looks good. How many PCR cycles you ran for this? Hope your PCR pre mixture is correct in order.
Junjie Shao I did not pre-heated the colony at 100oc since I thought the denaturing temperature of thermal cycler 95oc is enough for getting DNA from that. I just directly pick the colony and try to inoculate the bacteria straight to my PCR reaction, the total volume is 25uL.
Dinum Herath I ran 30 cycles for this and PCR mixture is good since the positive control worked.
I think the problem may be the picking since I thought the inoculation is easy for bacteria and I did not witness the colony drop in the reaction. I just pipetted several times and swirled several times. May this part be in trouble?
double the concentration of Amp of the LB plate, and spread less transformed E.coli to the plate (make sure to get single big colonies).
Shibo Bai the number of cycles are enough. true you just need to confirm you placed the colony properly in to the tube. better to put the colony first with sterile toothpick or pipette tip in to the bottom of the tube, then add your master mixture.
- Badges
- Science topic