I have actually some technical questions :
1. I want any one of you to send me a simple protocol for Poly A adenylation of miRNA followed by RT reaction for cDNA synthesis. I will give u one , if any comment, let me know
- polyadenylation in a reaction volum 20 µl
Poly A polymerase E 1 µl (5 unite)
Poly A polymerase E buffer (10 X) 2 µl
RNA templet (1 µl of 400 ng/µl)
ATPs ( 10 mM) 2 µl
water 14 µl
for 37 C for 10 min,
then I will measure again the RNA concentration to adjust it again to 400 ng / µl then I will prepare a RT reaction volum 20 µl
DNTPsa 1 µl
RT E 1 µl
RT E buffer 4 µl
RNA templet 1µl
oligo Dt primer 2 µl
Water 11 µl
mix the mixure, centrifuge it then 50 C for 30 min and 85 c for 5 min ,
Is this right or wrong ?
2. I received a ATP as a stock of 10 mM and as clear from above I want to use 1 mM in the reaction volum 20µl. How can I calculate the volum that I need to be added from this information
3. The Oligo dt primer that I received is 100 µM and I need about 100 picoM in the reaction volum 20 µl. How can I calculate the volum that I need to be added from this information ? I think the same answer like above.
4. Why there is a difference between primers used for q-PCR and normal PCR for the same gene ? I know that u have to dilute the primers 1 : 10 or 1 : 20 if used for q-PCR.