I am trying to generate a 2x mCherry sequence using overlap PCR. The first reaction generates good fragments with overhanging sequences (linkers) but the following 'fusion reaction' does not work. Any ideas why the second reaction does not work? It is important to say that the two fragments I want to fuse (mCherry sequence) have identical sequences apart from the designed overhanging linkers.
Here is what I do:
Primer 1: 5'-(25bp nesting region 1)ATGGTGAGCAAGGGCGAG-3'
Primer 2 (linker): 5'-(CCCGGCAAGGCCCGC)CTTGTACAGCTCGTCCATGC-3'
Primer 3 (linker): 5'-(GCGGGCCTTGCCGGG)ATGGTGAGCAAGGGCGAG-3'
Primer 4: 5'-CTTGTACAGCTCGTCCATGC(25bp nesting region 2)-3'
Primer 5 and 6: 25bp nesting regions in primer 1 and 2, respectively.
I generate two fragments using primer pairs 1+2 and 3+4. I purify these fragments and use it in a second reaction where I use nested primers (5+6) to amplify the larger fragment. This second reaction does not work.
This might reflect my misunderstanding the experiment but it sounds like the nested primers would be present in each of the two pieces to be fused together with fusion pcr... You get just the small band I am assuming? There should be a unique sequence added to primer 1 and 4s 5 prime ends, which can subsequently be used for fusion PCR.
Thank you Robert. The nesting regions are different. However, I only get the small band. I will try optimizing the melting temperature for this new reaction by gradient PCR. Maybe the optimum Tm changes?
How long are the overlapping sequences within your fragments, longer than the 15 bp linker? Of course you can try to optimize the annealing temperature but if the identical sequences are much longer than the linker I fear you will have no luck, even with this GC-rich linker.
According to the NEB cutter there are several restriction sites within you linker, any chance to try it with conventional cloning?
Thank you Michael. I have two equal sequences of 720bp in these two products (mCherry sequences). I want to create a duplicate mCherry protein (2x mCherry).
I guess I will have to change strategy then and design unique linker that can be cut by digestion. :-/
Yes, with this homology you have to change your strategy. Good luck with cloning!
Dear Jie,
I am testing the effect of different molecular weight (size) in my experiments. Hence the double mCherry. :-)
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