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cloning an AT rich gene


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3 replies to this topic

#1 nahlagadalla

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Posted 06 February 2012 - 08:17 AM

Hi,
I am new to cloning and facing a big problem.
I am trying to clone an AT (80%) rich gene from Plasmodium.
I have PCR amplified the target gene, extracted the band from the gel, purified it with Qiagen Gel extraction kit.

I run it on a gel to estimate the concentration and ligated it into PGem plasmid using the PGem Teasy kit in a 1:1 molar ratio (insert to vector).

Transformation looks o.k.
I did colony PCR with insert primers and got a strange size band, 1.2kb instead of 550bp? what could this be?

the other problem is when I try to sequence it with SP6 and T7 primers I only get the vector sequence.

did the ligation not work? how could I test it?


thanks

#2 scolix

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Posted 06 February 2012 - 10:01 AM

the ligation probably didn't work. that's why the vector sequence came up from sequencing.

how did the ligation plates look like? control vs ligation?

#3 Curtis

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Posted 06 February 2012 - 10:46 AM

I don't like colony PCR. it gives false results. Contamination of unligated DNA from your ligation reaction might also be there when you spread your bacteria on plate.

#4 HOYAJM

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Posted 06 February 2012 - 12:05 PM

If you are ligating the insert into the vector using restriction sites vs. blunt cloning, just do minipreps for some of the colonies and do a restriction digest to release the insert and then visualize that on a gel. I agree that colony PCR is very unreliable. I have observed the correct band from colony PCR for 10 colonies only to find out 8 of them were false positives.

Also, you could just linearize your ligation product using a RE with only 1 site and just observe the length




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