The Jacks Lab
protocols

miR-17~92

 

Reaction Mix (25ul):

Tail DNA 2.0ul
NTPs 0.5ul
10x PCR buffer 2.5ul
miR F4 0.1ul
miR R5 0.05ul
AV 145 0.05ul
milliQ 19.6ul
Taq (1/10) 0.2ul

 

10X PCR Buffer:

500 mM KCl
100mM Tris pH 8.3
15 mM MgCl2
1 mg/ml BSA
2 mM dNTPs
dH20

(store at -20 C)

 

PCR Primers: all used at 100um

miR F4 5' TCG AGT ATC TGA CAA TGT GG 3'
miR R5 5' TAG CCA GAA GTT CCA AAT TGG 3’
AV 145 5’ ATA GCC TGA AAC CAA CTG TGC 3’

PCR Conditions:

1. 94C 5:00
2. 94C 0:30
3. 55C 1:00
4. 72C 1:30
5. Goto 2 x34 cycles
6. 72C 5:00
7. 4C 0:00
END

Expected Bands:

WT 250bp

 

MUT FL

289BP

Mut KO

441 bp