This is a mission shRNA library retrieve PCR process. The given primer in the kit called LAP1 and LAP2 product is 342bp, which is too large to proceed the PE150 illumina sequencing. So I self-design a F2&R2 which yield product of 111bp. The attached is the 2% agarose gel pic, last five marker binds present 100, 200, 300, 400 and 500bp,respectively. The last lane in the cell round 2 pic showed the positive control which is also slightly smaller than the lung metastasis gDNA sample. The lung sample is confirmed with T vector, column purified. The sequence is exactly the one I want.
At first I did the round 2 by adding 1uL of round 1 product 100-1000 fold dilution.Then I tried Ampure beads and gel purification to purify the LAP product (round1),but it doesn't effect the bind size.
It seems like the problem is on it own. I'm doing the T vector ligation for the large bind now. Do anyone have the same problem or have some clues? Thanks a lot!!!
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