Transformation problem. Help me! - (Jun/19/2006 )

Hi,
I inserted a 580 fragment into TOPO 2.1 and transformation by using TOP10 competent cell.
After that, I ran colony PCR to choose clones containing my insert. But I got some problem.
- some white colonies have insert, some have not and some dark and light bue colonies have my insert.
- PCR products have many bands [/b] including my insert.

I tried to choose 100 colonies but there are just 30 colonies containing my insert.
I can not understand where wrong is.
So I expect to get your help as soon as possible,

if you have any iformation about my problems, please send me through email: vuthuhuong289@yahoo.com
Thanks.

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Small inserts often give blue or light blue colonies due to read through of the LacZ. You can get white colonies if you don't spread the X-Gal evenly on the plate or if you have primer dimers present in you PCR. Primer dimers can give you small inserts that look like empty vector.

Daniel

Automated DNA sequencing improvement

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thanks Danie,

But in the case PCR products have many bands including my insert. Is it my sample contaminated some thing?

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The mostly likely cause of these extra bands is PCR overcycling. With colony PCR you need to use far few number of PCR cycles than with genomic DNA (I generally use around 20 cycles).

Daniel

DNA sequencing protocols

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I already used 32cycles, I got some samples having standard band but others is unusual. So I can not explain why and How I have to improve. I tried to do 3 times.

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Try doing the PCR again using only 20 cycles - 32 cycles is too many for colony PCR.

Nucleic acids FAQs

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I already tried to run PCR with 25cycles. It is right I get two bands, but the efficiency is very low.
there is only 3 colonies containing my insert out of 15colonies.
Is transformation is not good?
Ah, my insert has three bands but just one strong bands.
If I have to run PCR again?

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