Hi,
I'm attempting to clone a gene into a shuttle vector that doesn't have any standard features for protein expression.
I designed primers to capture my gene (~1500bp). The forward primer has a 5' ribosome-binding site, and the reverse primer includes a 6His-tag so I can purify the protein. Both primers include conventional restriction sites for directional, ligation-based cloning. My first attempt worked great and I got a 1500bp product that ligated into the vector and sequenced correctly. I found the 6His-tag was detectable by Western blot but wasn't binding to Ni-NTA. This is a large membrane protein and it isn't unusual to see this, so I reordered the reverse primer, but this time it encodes a 10His tag.
Now, when I run PCR, I get a very faint band at ~1500bp and a very bright major product at ~750bp. I have boosted the stringency and ramped the annealing temperature up as high as I can, but the major product is always the 750bp size and I get little to none of the ~1500bp expected product. I have even used my linear product from the first primer set as a template and I still get this 750bp amplicon. There are no internal binding sites for the primer, I have BLAST searched my primer in case it's picking up contaminating E. coli genomic DNA, but nothing. When I switch back to the 6His primer set, the ~1500bp product is reproducible, even using the same template. The only difference between these PCRs is the expansion of a 6-His (GTGGTGGTGGTGGTGGTG) to a 10-His (GTGGTGGTGGTGGTGGTGGTGGTGGTGGTG) at the 5' end of the reverse primer. The 3' priming region and the 5' enzyme cut sites of both reverse primers are the same, and I use the same forward primer in both PCR reactions.
Has anyone seen this before? I wonder if the product is in fact the correct 1500bp size but is somehow forming secondary structures and running at a lower apparent size - maybe the additional tandem repeats at the 5' end of the reverse primer are causing this? This is a 1% TAE gel run about 20 mins at 120V. I'm attempting to digest and ligate this to see if it will clone, but in the meantime I'd appreciate any help interpreting this.
Thanks!
ACA
Simple fix: use your 6His tag ligated into the plasmid as the template and amplify with the Forward + 10His reverse primer.
That should give you the 1500 bp product and the longer His tag.
Good luck!
Hi Katie A S Burnette
This is exactly what I've done in the attached gel, second lane. The template was 10ng of the 6His plasmid using the same forward primer and the 10His reverse primer. I tried using the 6His PCR product at 10ng as the template too - same result as the second lane, major product of 750bp.
Edit: I used one reaction to gel-purify the two fragments, and the second reaction I column purified both bands. I digested each of these. Strangely, when I digest the column-purified products and re-run them on a gel, the 750bp band disappears and only a product at 1500bp remains. So, my PCR product is adopting some kind of secondary structure that causes it to resolve at 750bp. The same was not true for the gel-purified product of 750bp, which is odd. It must be resistant to digestion in the absence of the linear form that resolves to 1500bp?
You might be getting an unusually large primer artifact and not real amplification that results in the 750bp product. I've seen it happen with one primer I used to use. What do you see in your negative control?
Try using the plasmid as your template - do not digest it first. Or, use a rare cutter that you are certain will not cut in your insert to linearize. And include a negative control reaction.
Hi again and thanks for your responses.
No amplification in the negative control and no signs of primer dimerization in any of these reactions. I'll try your rare-cutter suggestion, we have a few enzymes that should work to give me a quick answer.
The product I column-purified and digested which then resolved to the correct ~1500bp size did ligate efficiently. I should have a clone to check and sequence soon too. I'll post the results for curiosity's sake, and in case it helps anyone else with this problem!
ACA
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