(1). FLCN cDNA primer sequences:
Forward primer: 5’-AGC CAC CCC CAG CTC TTC AG-3’
Reverse primer1: 5’-TCC TCG GTC GGA GCA CCT TC-3’
(2). PCR product size:
No band in UOK257 cells. One 501 bp band in UOK257-2 cells.
(1). Recipe for 15 ul reaction volume
DNA----------------------------1-2 ul (around 25- 100 ng )
10x PCR buffer------------------1.5 ul
50mM MgCl2-------------------0.6 ul
5 mM dNTPs-------------------1.5 ul
10 uM Forward primer----------0.5 ul
10 uM Reverse primer-----------0.5 ul
5 units/ul Taq polymerase--------0.1 to 0.2 ul
Add ddH2O to -----------------15 ul
(2). PCR reaction:
1). Denature DNA at 95°C for 5 minutes
2). PCR cycles:
94°C-------30 sec
60°C-------25 sec
72°C-------50 sec
For 35-40 cycles
3). Extension: 72°C for 10 minutes
4). Store at 4°C.
3. Runing agrose gel
