I've been trying to subclone a LoxP-DsRed-LoxP insert, and I'm having a lot of difficulty with the PCR. I played with the annealing temp and tried adding DMSO and High GC enhancer, but no luck.
Basically, I am able to get some amplification, but it is very weak, even with the DMSO or enhancer buffer. I was wondering if anyone has experience with this or if this is a more common problem when cloning loxP-gene-loxP because of the palindromic nature of those end sequences.
You said that you had a 'weak' band. You can try this: cut out the weak band, and gel-purify them. After that, use the eluant as template for another PCR. Better to use a hi-fi PCR polymerase. If you get a good single band, sequence it to make sure that everything is correct.
What kind of subcloning method you plan to use for cloning in this LoxP-DsRed-LoxP insert? Creating two unique restriction sites on the two ends by PCR for ligation? Or using Gibson Assembly method (ligation-free)? I think that the palindromic nature of loxP sequences can be a problem for PCR if you use them as priming template. I would avoid designing primers on the 2 loxP sites for PCR.
Re-PCR from the purified band resulted in a smear pattern, and the palindromic nature of the loxP ends makes the ligation-free methods similarly non-specific.
In the end I just bit the bullet and used a lot of my Q5 enzyme and did 10 reactions. After gel purification the purity was very low from all the gel that is included when you do it this way, but I was able to scrape together enough to proceed, although now I'm out of enzyme and my boss is complaining about the cost... XD
1. Due to the palindromic nature of primers (loxP sequences), I won't be surprise to see smear pattern from Re-PCR results. It is one of the predicted outcomes.
2. Yes, the ligation-free methods can be similarly non-specific, because you still need to PCR your fragment.
3. From your last paragraph of your last post, I have a question: do you know the 'weak band' is really what you want? A correct PCR product? Is the size correct? Have you sequenced them? Besides, a correct size doesn't warrant that the product is what you want.
4. If you want to generate the fragment by copying a plasmid sequence (which has -xxxxx-loxP-DsRed-loxP-xxxxx fragment), you can PCR it out with 'RS1-xxxxx' and 'xxxxx-RS2' primers (PCR product see below**), xxxxx represents a 15-mer unique plasmid backbone sequence. RS1 and RS2 are two added unique restriction sites for later cloning. Try not to include partial loxP sequence in either primer. This way might be less complicated compared to use primers containing the 2 loxP site sequence. The xxxxx sequences should not affect your downstream experiment to remove the DsRed gene flanked by loxP sites from Cre-mediated recombination.
** RS1-xxxxx-loxP-DsRed-loxP-xxxxx-RS2
My original primer design was xxxx-RS1-loxp-DsRed-loxP-RS2xxxxx.
I am unsure whether or not placing the mismatching bases on the 3' end of the primer would affect PCR amplification, and I have re-ordered primers similar to what you have recommended, but about 15 bp farther away from the LoxP sites to hopefully further reduce the chances of self-annealing.
The additional base pairs should not affect my downstream functioning, as it will be subcloned downstream of a CMV promoter and I checked to make sure there are no ATG sequences before or after Cre-recombination.
I have no way to confirm the 'weak' band I saw was in fact the correct sequence, as I did not get enough of it to sequence, and after ligation, transformation and antibiotic selection, none of the surviving colonies had the insert; only the original re-circularized vector. Due to the holiday weekend here, I have no received my new primers yet, so I don't know what results I will get.
Have you had a luck to get a product you want with those re-ordered primers?
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