Re: [DIYbio] Re: First full DNA extraction, PCR, and gel

Yeah next time around probably going to aim for 60s extensions, but
drop the anneal and denaturation to ~10-15s and try to use some glass
tubes as well as plastic. Also never did an initial long denaturation
step which I see sometimes used, or a final extension like you said.
I havn't messed with the interface and programming on the PCR machine
much but plan on reading over the old manual, was in a rush that night
to get everything done in one go. Just wanted to set a baseline of,
"ok this works at least" and then go from there as for fine tuning.

Just put them in the mail this morning at 10am for sequencing, forward
primer only, so we'll see how the data comes out.

As for primer dimers, could take a look at the concentrations but they
were the same for both primer pairs with 0.5 uL @ 10uM concenation.
Notice the ITS primers were the only one with dimers, the other pair
weren't as noticeable, if at all. So that might suggest it's
something besides concentration, but perhaps my friend messed up
calculation when he made them and they are at a higher concentration
then listed...or since I did the ITS primer set while he did the NS1
set, maybe I messed up somewhere along the line in how I setup the
reaction.

I have a PCR cleanup kit but, got 2 free sequences from GENEWIZ for
normal / custom order, so figured just go with custom and let them
clean it up and figure out concentration with their nanodrop or
whatever they use. This'll be the first time we'll ever get our own
data to use with BLAST, so hoping to learn more about the
bioinformatics side, I'm sure that's an entirely different beast.

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