RNA isolation, RT-PCR, and library preparation

RT-PCR recipe and conditions

cDNA synthesis:

8 µl        total of 1 µg of RNA

1 µl        50 µM Random Hexamer Mix (Invitrogen)

1 µl        10 mM ea dNTPs

65°C, 5 min

Ice, 5 min

4 µl H₂O 

4 µl 5X FS Buffer (Invitrogen)

1 µl 0.1M DTT

1 µl SuperScript III (Invitrogen)

25°C, 5 min

50°C, 60 min

70°C, 15 min

Ice.

PCR:

Dilute cDNA 1:5 into H₂O

10.9 µl H₂O

4 µl cDNA

2 µl 10X Taq Buffer*

1.6 µl 25 mM MgCl₂

0.4 µl 20 µM Forward Primer**

0.4 µl 20 µM Reverse Primer**

0.2 µl 10 mM ea dNTPs

0.5 µl Taq DNA pol.

Cycle conditions:

1              95°C       0:30

2              95°C       0:10

3              57°C       0:20

4              72°C       0:20

5              back to step 2 (36 cycles for CEP131, 35 cycles for CNTROB, 30 cycles for Actin)

6              72°C       7:00

7              4°C         hold

* 10X Taq Buffer:

500 mM KCl

100 mM Tris pH 8.5

0.1% Triton X-100

** Primer pairs:

ACTB-F: AGAGCTACGAGCTGCCTGAC; ACTB-R: AGCACTGTGTTGGCGTACAG; 

CNTROB-4F: TGCAA­GACTTGTCTCCATCTAGCTC; CNTROB-5R: TTGTCCAGTTGTTC­AATCATGGTATCTTTC; 

CNTROB-9F: AGAAGAGCCAGAGGG­AAGCC; CNTROB-10R: TTGCCGTAGGCTGCTCTCC;

CEP131-4F: ACGGAGCCCACAGACTTCC; CEP131-6R: CGCAGTTGCCC­ACTGCTC;

CEP131-14F: TGGGGTCCGAGGTGAGC; CEP131-15R: GCTGGATGGTGGCCTCG