biologg

Hi all,

a quick question: I have sequence A and sequence B, which i would like to fuse together, so that i get a product A-B (fragment A is aroudn 500bp and fragment B around 1200bp), which i will then clone. I have been told that the easiest thing to do is overlapping sequence PCR. My question is, how many nucleotides of overlap are sufficient for both fragments to be fused? If I design "overlapping" primers of around 20nt of length, can I intrtoduce 5nt sequence on the 5' end of each of these primers, which would be complementary to a sequence of the other region? So overall, the overlap will be 10nucleotides. Will this be enough for the fusion of the two fragments to occu? Or should the overlap region be bigger? I am not really sure how many non-complementary nucleotides i can introduce to the 5'end of a primer without seriously affecting its template annealing capability.

Hello all,

I have been struggling in the last couple of weeks to amplify 2 sequences from cell isolated gDNA (both sequences are between 75-80% GC rich; 700-900bp long). I had lots of problems with unspecificity with both Taq polymerase and Phusion polymerases. I have tried 3-5% DMSO, GC buffer, temperature gradient for the annealing temperature of the primers, two sets of primers...non of these worked and I got lots of unspecific bands, but none of the expected size of my amplicons. Then, last week i tried Taq+2M Betain and it worked like magic! I was really impressed by the difference in specificity between Taq alone and Taq+Betaine and both sets of primers work fine with both sequences. So far so good, but I tried 2M betaine with Phusion polymerase and it was the same mess as without betaine - lots of unspecific bands. I need the amplicons for sequencing, so i would prefer to use a high fidelity DNA polymerase.  Has anyone experienced a similar problem? Do you have any suggestions how to improve the specificity with Phusion? Is unspecificity a common problem with high fidelity polymerases in general, or should i try another polymerase, such as Pfu? Alternatively, i guess i could just take several replicates and do the sequencing with Taq - it would be highly unlikely that Taq would incorporate the wrong nucleotide in exactly the same location between 4-5 replicates. But I am also curious to understand why would high fidelity polymerases be more unspecific than TAQ?

Hello everyone,

i have always been wondering something, but never quite got a definite answer. Many lysis buffer recipes for DNA extraction contain both Prot K and RNAse. However, it makes no sense to me to add both at the same time, as Prot K degrades RNAse as well, does it not? So what is the point to add RNAse to get rid of your RNA, when Prot K is also present in the reaction? Does it make any difference if you add the RNAse just before you add the lysis buffer to your cells/sample? Maybe in this way the RNAse will degrade the RNA molecules (or at least a large amount of them) before the Prot K had the chance to degrade the RNAse? Some suggest adding RNAse first, but if you add it before the Prot K, then you risk DNA digestion by DNAses. Can someone clarify this for me? Thank you!

Hello,

I would like to ask you the following: I need to extract genomic DNA from 2 different cell types, run a PCR to amplify an approximately 900bp fragment and send the PCR amplicon for sequencing.  Can someone please tell me, as I am quite inexperienced in this:

After genomic DNA extraction, what would be the best way to quantify the amount/concentration of DNA I have, in order to load it on the PCR plate? Can I use nanodrop, for example? Someone told me nanodrop is not a good idea, as following extraction, my samples will contain lots of RNA and i will not get correct measurement. But this maybe depends on the protocol for genomic DNA extraction? Can you recommend me a protocol?

Overall, this is what i would like to do:

1) extract genomic DNA from cells
2) determine DNA concentration
3) run PCR to amplify sequence
4) run the product on a gel and purify it
5) send for sequencing

I would appreciate it if you can give me some advice or if you think i should do some additional steps.

Thank you!