Hi,
I need to clone 30-40 PCR products. I tried direct sequencing of the products but found out that they are mixed and result in overlapping peaks if directly sequenced. I want to clone these products so I can sequence 4-8 resulting colonies.
I find it tedious and boring to do single column PCR extractions of the products prior to ligation. Any suggestions to avoid the PCR clean up step and directly clone them into a vector, and to avoid that I clone the primers (exosap step needed)? Also, any good suggestions how to plate the resulting transformations? I would only need up to 10 colonies per ligation. 30-40 plates is a lot... I only have access to Sanger sequencing, not to NGS.
I was considering trying various dilution steps of the transformation so I can plate more than one transformation on a plate. Was thinking undiluted transformation, 1:10 dilution, 1:100 dilution and then drop 20 uL or so on a designated spot on a plate so I can spot more than one transformation event on a single plate. Then next day, pick the colonies.
Thank you in advance for your suggestions!
Natascha
I have frequently just cleaned up PCR products with an ethanol precipitation. There will still be some primer that comes along but that normally does not interfere. However it may depend upon how you intend to do the cloning, restriction digestion, TA cloning, blunt end cloning. I would go ahead and use 30-40 plates, the possibility of cross contamination is too high. My biggest concern would be ensuring that your clones actually have an insert.
PCR cloning kits allow you to insert PCR products in different vectors in a very convenient and easy way, with no purification needed. You should still run 5-10ul of your PCR products on a gel to make sure the amplification was specific. If using a blunting polymerase such as Phusion, the ligation step only takes 5mins.
These kits usually result in hundreds/thousands of colonies from which >90% have the correct insert. I suppose you can dilute your transformation mix 1:100 and spread 10 uL spots.
OR you could ask the sequencing company to take care of the purification step ;) It's barely more expansive than the sequencing itself.
I would follow Michael Benedik's recommendation about the plates, unless you have an absolute shortage of plates. However, if your are very sure of the efficiency of your cloning and transformation procedure, you could draw 4-6 sectors on each plate, spot a few µl of the transformation mixture on each sector and streak them with a loop just as you would do to isolate single colonies
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