Hello everyone,
I had multiple PCR products after doing the colony PCR on white colonies (primers SP6-T7, annealing temp 55 degrees) which are supposed to contain only one insert. I cloned a bacterial 16S (about 1500 bp), into a Promega pGEM vector (about 3000 bp). The previous analysis about the 16S cloned insert (gel visualization, purification, concentration measurement) were ok. The cloning procedure was ok (as usual).
As you can see in the photo, in lane 4, 5 and 8, besides the 1500 bp insert there is a smaller one at 1000 bp and a bigger insert above 3000 bp.In lane 8 the bigger inserts were three... (The marker is Generuler 1 kb)
I have some ideas about the reason but I'm curious to know your experience about this.
Thank you for your replies
Cristina
I am not 100% sure, but it looks to me that the bands at 1000 bp and 3000 bp could be circular plasmid (CCC and OC).
Another possibility is that your transformants have several different plasmids. In that case, you can re-streak your transformants to single colonies, and examining couple of them by colony PCR again.
I think 3000 bp band might originated from circular plasmid, just like Mr. Hiraga said. As for 1000 bp, maybe it is from non selective binding during PCR? Maybe you can try increasing annealing temperature or adding DMSO to the PCR reaction to remove the extra band.
This might be due to the non-specific binding of the primers to the genomic DNA. You can try gradient PCR with different temperature.
Too little information to be very helpful.
What PCR primers did you use for colony PCR?
Multiple inserts will still generate white colonies! What was the cloning strategy?
Smaller inserts are more difficult to explain!
Primers were the one flanking the insert in the pGEM plasmid, SP6-T7, the annealing temperature was 55 degrees.
The cloning strategy as the manufacturer instructions (Promega Easy pGEM), as I did for thousands times without any problem...
I actually thought that the bigger one is the circular plasmid, but I was wondering how it is possible -.-
For the short fragment yeah, is really difficult to explain!
PCR products? RE products? Blunt ends? Sticky ends? Same ends? Different ends?
Multiple inserts are the easiest answer to large fragments.
One thousand constructs deserves a prize!!
There could be many reasons..
1- Which Taq polymerase you are using for 16S PCR and the number of cycles you used for amplification??
2- Did you pool PCR product and then purify it?
3- What was initial denaturation time for rapturing cells?
4- What was extension time for colony PCR ?
5- You can try M13F and M13R primers also..
There could be may reasons..and this is the problem of PCR not cloning..just you have to spend few more days to standardized it..
Geoff, if they were re-products or blunt ends for sure I have write it...It 's a 16S, simply. The Promega kit is the most used in all laboratories, the procedure is always the same. Thank you!
Sanjay, the Taq is an Hot start Go-Taq from Promega, the initial temp was 96 degrees, the extension time for each cycle was 1.30 mins and the final eleongation 5 mins. Yes, I think I will try with the M13/22 mer primer and for sure I will modify some parameters in the PCR conditions, thank you!
dear ma'am,
all wells from different colonies? it would be
I am not sure probably it could be contamination to your reagents.
and try to give some more time and that is for gel.
it could also be as a result of the quantity of sample been amplified before loading of the gel. for example if the amount of DNA used is more, it could produce some bands that are not very visible apart from the desired bands... i observe this recently and had to perform some more dilution
Hello! did you manage to get over this problem in the end? Can you give me some feedback: i have a very similar issue (with a second shorter band of 500 bp for a 1000bp expected fragment).
best
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