surprising behavior of transformants needs immediate advice!
Posted 24 October 2012 - 06:29 AM
I have been trying to clone a 3Kb pcr product into a TOPO vector. I have tried the standard protocol given with my vector kit many a times. This protocol includes:
1. Pcr amplification of desired gene with phusion (NEB)
2. gel extraction using qiagen kit
3. addition of "A" using taq polymerase (openwetware protocol) at 72 degree for 20 mins
4. ligation using TOPO vector for 30 mins at RT
5. transformation using TOP10 competent cells at 42 for 45 secs.
6. addition of 250ul SOC to this mixture, incubating shaking at 37/1hour
7. plating the transformants on LA+Amp.
I tried this procedure many a times and got very small satellite colonies. These colonies very small and did not grow in LB + Amp.
One fine day i followed the same procedure and again got small colonies like before. I picked few of these small colonies and grew them in LB+Amp for 4-5 hours. As expected, there was no growth. BUT after one day ie after 24 hours there was growth in this LB+Amp and also on my plate (LA+Amp) the small colonies had increased in size. Initially i thought that this must be some contamination. So to test this, i innoculated 15ml LB+AMP with small innoculum. I got growth in 2 hours.
I am planning to isolate plasmid from these cells and check for my clone. BUT before i do that i need an expert advice on the possibility of these colonies being the desired transformants.
KIndly help and please reply as soon as possible
Posted 24 October 2012 - 06:33 AM
Posted 24 October 2012 - 12:42 PM
It could also be that your transformation protocol is not the most efficient - it is quite dependent on the tubes that you are using for the heat shock step as to how long you should shock for. Using eppendorf 1.5 ml tubes I shock for 1 min.
It is also possible that the addition of the A overhang is being inhibited by some carry-over from the gel extraction - you shouldn't need to do a gel extract if your PCR is specific enough.
Satellite colonies are colonies found around a main colony on a plate, small colonies are just small colonies...
Posted 24 October 2012 - 11:00 PM
Thanks for your reply. I isolated plasmid from these small colonies grown in broth + amp. it gave me a concentration of 0.1ug/ml. A260 = 0.001 and A280 = -0.003. I think this is not the plasmid.
The kit is very new and i have no idea as to how topoisomerase could go off? I get many bands in pcr which is why i have to do gel extraction of my desired band. I have even tried to give heat shock for 1 min but still no colonies.
I am unable to troubleshoot as to where the problem is?
Posted 25 October 2012 - 12:44 PM
How have you optimised your PCR? You should at least be able to get it down to a few bands with the right conditions, unless your primers are particularly short.
Do you have a positive control reaction for the transformation?
Topoisomerase, like any other enzyme can easily be degraded by temperature and freeze/thaw cycles. Follow the kit storage instructions, and make sure you keep the tubes on ice when thawed.
If you can post a complete protocol for all your steps, that will help diagnose the problem - it is very likely a simple mistake that you are making.
Posted 25 October 2012 - 02:10 PM
Posted 25 October 2012 - 10:15 PM
Fwd: 5’ ACATGCTTTGGGACTGCCACTGA 3’
Rev: 5’ GCCGGCAATGGACGTGAACA 3’
I am using 1x buffer, 25mM mgcl2, 1.25ul primers, 0.25mM dntp, 1U/ul Taq, 1ul template for a 25ul reaction
Posted 25 October 2012 - 11:26 PM
You primers probably have quite different Tm's as they are 70 and 64 respectively. You might be best off designing new primers to make the PCR more specific.
Posted 26 October 2012 - 01:42 PM
Also, what PCR program are you using?