I performed PCR on genomic DNA with gene region specific primers that have a M13 adapter, which resulted in two PCR products. I treated the PCR products with ExoSap (primer removal) and cloned into vector pMiniT (NEB). Single colonies were subjected to colony PCR using M13F and M13R primers. It resulted either in single (lane 1 bigger, 14 smaller) or double bands (lane 2, 5) where I expect to see only single products.
M13 primers do not bind in the vector (I checked the sequence by BLAST and Clone manager) and therefore can only amplify from the fragment.
Why am I seeing two fragments from some of the colonies? Its 25 cycles at 50°C annealing.
Could some of the colonies have two different plasmids, some of them with loss of sequence due to homologous recombination within the cloned fragment?
I forgot to mention that the negative control (no DNA added) was clear, no background amplification.
It may be due to
1) the colony contain more than 1 type of plasmid (i.e. with inserts and without inserts). At first i thought this should be rare, but after I work on my project, i found this is very common.
You should avoid colonies like this (while you have both single and double bands, ignore the double band and take the colonies with expected single bands) . Because when the bacteria propagates, the "unfit" plasmid construct will lost.
you can read this: http://bitesizebio.com/2267/plasmid-retention/
2) multiple copies of inserts. Your plasmid may contain or than one copy of insert (the colony)
3) non specific binding to genomic DNA. Could be check by PCR of the purified plasmid products.
4) Two vector binds together, without inserts. To reduce the change of this happens, increase the molar ratio of your insert.
Good luck
When looking to your gel there are more than 10 line where you have a double band and three with a single band. I think tha you have a problem with the PCR or the quantity of bacteria used for the PCR and what you are showing is the different form of the plasmid. What is the size of the fragments you expecting? that will help if they are close to one form of the plasmid.
Hi, i think the problem is M13 primers you can check it by T7 , and if it doesn't work there may be a problem about direct adding of bacteria to your reaction and you should use a filtered pipet tip to pipet up the bacteria, i think there is some contamination in your PCR i hope it works.
I think you have multiple inserts in the same plasmid. You can do plasmid extraction from each kind of colony and then cut using restriction enzymes outside the insert cloning sites. The size difference will tell you if you have more than one insert , no insert or one insert. Just make sure your insert does not contain the restriction enzymes you use for digestion.
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