Cloning PCR product - double bands or heterozygote (Oct/16/2005 )
Hello. I already got the target band from my PCR. I'm doing microsatellites. How will I purify double bands (heterozygotes) and can I clone the PCR product directly to a vector knowing that I have double bands or heterozygotes?
I am a neophyte at cloning.
I am a neophyte at cloning.
Yes you can clone this band in any convenient vector I would recomment pGEM-T or pCR Topo vector. They will clone easily pCR bands which A overhangs (added by the Taq during amplification).
Success also depends of the size of the bands what are the size of your inserts ?
For purification they are dozens of possibilities depending of the size of your product !
Pesji
Hi Pesji,
I noticed that on the gel when I viewed my PCR product, I got 2 bands for some samples, 1 on others. I dont have any problem on single bands. I clone them successfully. But when I purify the double bands (which are very close to each other), I only got single band. Where did the other band go? My target band is 350bp and on heterozygotes, there's an additional band either below or above it. They are very close, it's hard to excise from the gel the 2 bands separately.
Can I clone directly the 2 bands in PGEM-T easy without purifying it, coz when I purify, the other band is lost.
Thanks for the info.
Jedi
I noticed that on the gel when I viewed my PCR product, I got 2 bands for some samples, 1 on others. I dont have any problem on single bands. I clone them successfully. But when I purify the double bands (which are very close to each other), I only got single band. Where did the other band go? My target band is 350bp and on heterozygotes, there's an additional band either below or above it. They are very close, it's hard to excise from the gel the 2 bands separately.
Can I clone directly the 2 bands in PGEM-T easy without purifying it, coz when I purify, the other band is lost.
Thanks for the info.
Jedi
Yes you can clone both at the same time and let the bacteria do the selection for you you do a pCR screening of the colonies and you pick 3 or 4 of each insert
As for purification when bands are so close it's rather difficult to separate them !How different are they in size 10, 50 100 bp ?
Which percentage of agarose do you use for your gel ? Which buffer TAE or TBE
pesji
I use 1.5% agarose gel on TBE. The difference of some double bands are 50bp and below. I tried running the gel a lot longer but bands are so close it's hard to separate.
Ok, I'l try to do what you suggest. It's a great help.
Many Thanks...
Ok, I'l try to do what you suggest. It's a great help.
Many Thanks...
For this difference in size it's too difficult to separate for real efficient purification you will always get some contamination of one bad from the other so I guess it's far easier to let the bacteria do the job
Wish you sucess !
pesji