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Subcloning


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#1 taylor

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Posted 29 September 2009 - 06:35 PM

Hi I am new to molecular biology. My question is once I do the ligation of the vector and the DNA of interest can I do gel purification and then perform PCR to screen for a recombinant. Or is it necessary to do transformation, miniprep and then check for the insert. My vector does not have a LacZ. Thank you for your help:)

#2 fishdoc

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Posted 29 September 2009 - 07:21 PM

Hi I am new to molecular biology. My question is once I do the ligation of the vector and the DNA of interest can I do gel purification and then perform PCR to screen for a recombinant. Or is it necessary to do transformation, miniprep and then check for the insert. My vector does not have a LacZ. Thank you for your help:)



You can use the ligation product directly for template in a PCR reaction without purification, assuming you have primers just outside the cloning site. I can usually get a product from 1 ul of the ligation, but you may want to also do 3 and 5 ul, just to be sure.

All that does, though, is ensure your ligation works. The ligation working doesn't really matter if the transformation doesn't work, and when you transform, there's a possibility of something else getting in there, so you still need to verify the clones. Since you don't have lacZ, you can do some pretty rapid screening by colony PCR. You'd use the same primers you plan to use for the PCR of the ligation. For colony PCR, I take a small portion of a single colony on a 200 ul pipette tip and spin it around in 25-50 ul of water, then use 1 ul of that as template for PCR. I've been successful amplifying inserts up to 5 kb using T3/T7 primers.

#3 taylor

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Posted 30 September 2009 - 06:35 AM

Thank you veteran,

So by doing sequencing PCR i will make sure that i have the insert and do the transformation with that DNA, so if I obtain colonies after transformation all of them should have the construct, is that correct? Thanks for your help

#4 fishdoc

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Posted 30 September 2009 - 08:14 AM

Thank you veteran,

So by doing sequencing PCR i will make sure that i have the insert and do the transformation with that DNA, so if I obtain colonies after transformation all of them should have the construct, is that correct? Thanks for your help



I wouldn't sequence until after transformation.




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