always two bands in colony PCR - What the hell did another band come from? (Mar/23/2008 )

Hello, there

I had a problem in confirming a insert in colony PCR. I had no idea why I always got two bands when I use PCR to confirm a insert.
I have been using "PCR-select cDNA Subtraction Kit" to build up a cDNA library. I had done the cDNA subtraction. Now I am using the TOPO 2.1 cloning kit from Invitrogen to clone the subtracted cDNA pool . I had a problem to confirm the cDNA insert after I had ligated cDNA into a TOPO 2.1 vector and picked the white colony and allow it grow in LB broth overnight. I used colony PCR to confirm a insert. My problem is that I always got two bands from my colony PCR no matter what primer set I use, e.g. M13 Forward and Reverse, M13 forward or reverse and one of my cDNA specific primers. The two bands always went together, the lower band was always about 200bp smaller than the bigger band no matter what the bigger band was. Because I am using a pool of cDNA (300-2000bp), I could not gel purify them. I also could not purify the cDNA pool with chloroform extraction, that way I will lose some of my precious cDNA (each cDNA in the pool stands for a differentially expressed gene between two tissues). Since the cDNAs contain EcoR I restriction sites, I could not use restriction enzyme digestion to confirm a insert either. I tried hard to optimize my PCR condition and rule out the contamination. However so far I did not find that the problem was from the contaminations of water, primers or poor PCR condition ( I optimized anealing temperature, concentration of template, primers and number of cycles for PCR).

Did anyone ever had a problem like this? What the hell was the extra band come from? is it possible that M13 primers can aneal twice on the insert? I really appreciate your help!


Eastern rabbit

I don't know the answer, but I can tell you how to find out. Run a PCR for your sample on a gel (large volume) and gel isolate the two fragments. Sequence them.

I presume you've tried raising the annealing temperature. You may be using too many cells in the colony PCR. Virtually everyone does. Touch a 10 ul tip to the colony (just touch it). Scrape the tip around the outside of a small tube with 50 ul water. Use 1 ul of the water in your 10ul PCR reaction. Keep the 50ul water sample in the refrigerator pending PCR results, or inoculate 2ul of the water on a master gridded plate.

Thanks for your reply!
My boss thought it sensable to sequence it only when you've got one band. I tried diluting the template 10X to do PCR and got the same results. I tried to increase the anealing temperature of the primer. Still the same results. My boss thought the extra band was a contamination, but how come a contamination gave a band with a variable size? The only reason I could think is the mispriming. Did anyone ever get two bands in your colony PCR? Or is it possible there was a contimination from the LB broth?

Hi,

Why don't you try to PCR with the nested primers provided with the kit? You can order the primers with the sequences provided in the manual.

If you're interested to know only the presence of insert in the clones (instead of the size of the inserts), another approach might be to do a colony lifting and subsequent hybridization with labelled probe (e.g. the nested primer as probe labelled with ECL kit).

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