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PCR Ligation cloning


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#1 shilpamadhu

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Posted 24 January 2005 - 07:07 AM

hi,
I am trying to clone 3 bits generated by pcr, and all three are nearly 0.8kb, one of which is chloramphenicol gene. I need to clone the other 2 bits flanking the antibiotic gene at either ends.
I have tried,
1. ligating all three bits at once,.... too many bands ( expected 2.4 kb)

2. ligated Ist bit with antibiotic gene, and same way with other one ( expected 1.6kb each). Although I got 1.6 kb, but when I checked by pcr with fwd primer of Ist bit and Rv primer of antibiotic gene, I did not get any 1.6 kb product. So it looked like similar bits were ligating.
I have also changed the REN sites, but no use.

Could someone help me please. I've been trying this fro past 3 months, but no result.

thanks.
shilpa :(

#2 george@CASE

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Posted 28 January 2005 - 12:41 PM

it sounds like one (or more) of your restriction enzymes aren't cutting.

I would suggest subcloning each of the 3 PCR products in pGEM-T Easy or some other vector that would immediately accept PCR products. Then, cut the PCR fragment out from the vector using your restriction enzymes. This way, when you isolate the 0.8Kb band from the gel, you know that both ends have been cut properly.

Ligation should be straight forward.

Also, take note that different enzymes can produce compatible ends which are just as efficiently ligated.

#3 shilpamadhu

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Posted 31 January 2005 - 03:11 AM

Thanks very much.
I'll try that.

shilpa




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