4 replies to this topic

[losybelle]

Hi:

Does anyone know about any vector which use U6 promoter?

Thank you

[Evanescence]

hi,
I dont know but may i know why u use U6 promoter? is that any particular reason?

[losybelle]

hi,
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...

[Evanescence]

i guess the problem is PCR problem...it's better for now for you to consider to re do pcr with several changes...i guess there's a vector with U6 promoter but it's hardly to reach...so for optimize your PCR

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:)
Cheers always

Evanescence

[losybelle]

thanks!