straight pcr to sequencing was not working out.
looked like it was going to be a huge problem getting the samples to be pure product.
with every increasing pcr (hoping that pcr of a pcr would start to purify it) made more primer dimers.
even getting product on the first try was flaky work.
finally decided to change the protocol.
on saturday, i got fresh buccal samples (me, becky and ing).
then i lyse-and-go the cells open.
then i pcr the samples.
i take the samples and ligate them to a vector (invitrogen ta cloning kit!)
then i take the vector (including the keratin gene insert, now ligated to the plasmid vector) and transform into bacterial competent cells (E. coli... which i also made ahead of time).
i grow them for an hour in luria broth.
then i plate it on tsa with ampicillin and xgal.
this is where i stopped.
the plates are in the 37º incubator.
if the colonies grow, they are amp resistant and have the plasmid in it. (goood!)
if these colonies are white they have the keratin insert in it. (good!)
if the colonies are blue, they do not have the keratin insert in it. (bad!)
if no colonies grow, they might not be ampicillin resistant meaning they don't have the plasmid in it. (bad!)
if it's good news, then i "dirty" pcr it, and miniprep the keratin and take it to sequencing.
if it's bad news, i might give it one more try, depending on what type of bad news it is (lol).
otherwise, back to the drawing board.
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