Posted 20 June 2011 - 07:47 AM
Does anyone know about any vector which use U6 promoter?
Posted 20 June 2011 - 09:05 AM
I dont know but may i know why u use U6 promoter? is that any particular reason?
Posted 20 June 2011 - 09:59 AM
Yes I wanted to use it as a cassette for mirna expression. That means u6 promoter mirna loop mirna and Ter
I already have the primers and i was using DNA to amplified but PCR is not working, and i was wondering if maybe using a vector with the U6 promoter as template my primers would bind there easily...
Posted 20 June 2011 - 07:11 PM
1. maybe you need extend your initial denaturation time..maybe if the site contains high GC percentage it will hard to denature since the G-C interaction is really strong:) so maybe you can make it 3min-4min at initial stage
2.Annealing temperature also important, try to play around the range of this temperature...maybe you should start around 50-58 if no band get it lower...try gradient pcr...if you not confidence about the range try Touch Down PCR
3.make sure you have suitable extension time, 1000kb/min...if your template is quite good and strong, 30cycle is enough of not maybe need 35..but as you go higher the multiple band will be obtained
dont forget check your primers, sometimes i face this problem but if you change the template you may also need to change the primers if necessary...but after all optimizing pcr is better coz after this you will pcr same thing many times...dont rush to get it we may miss something to learn my friend:)
Keep bright to try:)