Is there a faster method to screen for positive colonies besides picking single colonies, growing them and then sequence/pcr them for the insert?
Faster method to screeen for positive colonies.
Posted 27 April 2015 - 12:25 AM
Posted 27 April 2015 - 03:19 AM
If the insert is big enough, you can also try cracking lysis.
One important step is to centrifuge the debris, otherwise loading the colony on a gell will get messy.
Posted 27 April 2015 - 08:05 AM
I have a love/hate relationship with colony PCR. Sometimes it works, sometimes not in my hands. Too many false negatives for me to consider it a reliable assay, even when done side-by-side with working positive controls (purified plasmid). Just my opinion tho.
Bob- why does dipping tip in water help?
Edited by labtastic, 27 April 2015 - 11:19 AM.
Posted 27 April 2015 - 09:11 AM
The usual problem with colony pcr is far too many cells. My standard process is to pick colonies with a 20 ul tip on a pipettor set for 2 ul. I swirl the pipet in 50 ul of water in a 96 well plate or pcr tube strip, mixing and scraping against the side of the tube. The same tip is then used to pick up 2 ul of the liquid, which is then spotted onto a master plate (I usually use SBS format petri dishes, so the spots are also at 9 mm spacing). Then, I use a multichannel pipet to pick up 1 ul of the diluted in water samples to add to 9 ul of a pcr master mix already containing primers. Initial denaturation is extended to 5 minutes, then cycled normally.
Some things that can go wrong include mixed colonies, and, more seriously, excess DNA from the ligation reaction spread on the initial plate, which can then be amplified.
Posted 28 April 2015 - 10:22 AM
I sometimes did have problems with Colony PCR, but dilluting is important. Also using PCR primers where one is on the plasmid backbone and the other one inside the insert (or after the insert, then you would see band differences). Colony PCR is teoretically not much useful for very large plasmids, on the other hand longPCR can be optimized even on colonies, it that is to be a routine job.
"Cracking" on the other hand never quite worked for me, first of all is more laborous and hands-on-time-consuming (especially for 60 colonies or so) and at the end, the plasmid often had various forms so I was able to tell absolutely nothing (doesn't happen with all but when I needed to know if my plasmid didn't recombine.. It didn't help much).
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Posted 29 April 2015 - 09:55 AM
Well, cracking lysis works well when you're looking for a ~3kbp vector + 500bp insert, and you can run a couple truly negative colonies for comparison. If this is the case, then it's actually pretty easy and reliable in my hands.
Also, I don't use it alone if I have to check colonies after PCR cloning; seeing sometimes a multiplicity of PCR products with similar, but not identical size is valuable info you get with colony PCR but not with cracking lysis.
Funny though, because to me it's so simple that I use it to screen colonies in the rare cases when I expect very few of them to be positive (PCR is more expensive, my lab isn't a rich one).