My gene of interest is 91bp. I cloned the PCR product in to Pgemt vector and digested with 2 enzymes. I am getting blue white colonies. While screening the white colonies, I am able to get the Pgemt band after restriction digestion. But I can't see my band, which I want to elute.
Dear Kannan,
Pls load your gel picture and one thing you can do after checking restriction sites on used vector, you can reduce incubation time for restriction enzymes....
I cloned it in Pgemt vector...incubation time of my restriction is 2 hrs for 50micro liter restriction reaction...
my problem is that my gene of interest size is just 91bp....i think 91 bp cant be visualized using Etbr... in agarose gel..
I guess there may be 3 possibilities.
1. The plasmid concentration you use for restriction digestion is not enough. You can see the band of the plasmid backbone. But 91 bp is too small to bind enough EB dye unless the amount is high enough.
2. The agrose gel concentration is not high enough. For a 91 bp fragment, 2% agrose is necessary.
3. The colony you pick is false positive. This is also possible although you got white colonies.
Hope this can help you.
For a 91bp fragment, you may not be able to see the band if you use say like 1% agarose. You can use 2% agarose and also at the same time, you can try to PCR amplify your target. 91bp is also small, so use plasmid concentration in the scale of micrograms/microliter
You can resolve the bands on 3% Metaphor agarose of 10-12% PAGE gels.
Eluting a 91 bp restriction product from a PAGE gel is much easier.
Good luck.
As John mentioned, with a 91 bp fragment in a 3000 bp backbone (pGEM-T), you need to digest lots of plasmid in order to have enough of your fragment to visualize on a gel. If you are trying to elute your band for subcloning, you will need to digest even more plasmid -- you will have to digest 6 micrograms of plasmid to get 200 ng of fragment, assuming you have 100% recovery from your gel.
In this situation, I would instead PCR amplify the 91 bp fragment with the appropriate restriction enzyme sites on the primers, digest, and ligate into your target vector.
Thankyou all... i am using 2% agarose only...i will increase the restriction volume and do..if not i will proceed with SDS page.
Dear Kannan,
I agreed with Yuheng Lin and Andrew Tai. Make sure that you did not pick the false colonies. You may perform a colony PCR to ensure that you are going with a real positive clone as suggested by Pia Hermanns.
Next, you may have to start you digestion with more recombinant pGEM-GENE construct, to end up with sufficient target fragment after digestion and gel purification.
Otherwise, you may PCR amplify your target gene with oligos appended with corresponding site and directly clone into the vector of interest.
Good luck.
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