AAAAAAAAAAnd one more. Right now your primers are at 1uM final conc I believe. Try dropping that in half.
Do things stepwise, or vary only one thing at a time, if you throw in 2 or 3 changes into one reaction and it works, you don't know why it works.
Also, are your gels being run in a Tupperware container or something? I'd get new DNA ladder. What % gel are you running and what are you using to run it?
Your gels are smiling and smearing, so either your gel is getting to hot because it's to high a voltage, or you have a crappy power supply.
What size is your product again?
What did you put in each of those wells?
On Sat, Mar 5, 2016 at 8:00 PM, Dakota Hamill <dkotes@gmail.com> wrote:
Do that as well as a 2nd run with 1uL DMSO in a 25uL reaction.Sorry for the spamOn Sat, Mar 5, 2016 at 7:58 PM, Dakota Hamill <dkotes@gmail.com> wrote:Shot in the dark but drop annealing to 47c for 30 seconds, and increase denature to 98 for 45
On Mar 5, 2016 7:57 PM, "Dakota Hamill" <dkotes@gmail.com> wrote:That's an old guide but there are many out there like it. You could drop your annealing even lower. To,bad you don't have a second set of primers that you could use as a control on pVIB
On Mar 5, 2016 7:51 PM, "Dakota Hamill" <dkotes@gmail.com> wrote:I havn't used it in PCR in a while. I'm sure NEB, Google, or someone else on here could give you a more specific concentration. It can help with tough PCR.On Sat, Mar 5, 2016 at 7:42 PM, David Ishee <midgardkennels@gmail.com> wrote:What does DMSO do for it I have some. How much would I use?
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