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Mark’s RNAi insert size checking protocol using PCR SuperMix HiFi kit (Invitrogen P/N 10790-020).



Before: Grow up O/N LB Carb cultures of RNAi library clones in 37C shaker. for my stocks, to each 200ml LB bottle add 400ul 5mg.ml tetracycline stock in EtOH, 80ul of stock 100mM Carbenicillin. Aliquot 5ml per 15ml culture tube, inoculate, shake for <= 18h.



PCR Reaction using Supermix kit:



0. Did you sign up for a PCR machine with a small tubes block in it?



1. Mix ingredients in small 8-tube strips, plus one no template neg ctrl. - note clones/order, lot# of supermix.


Vol. in ul

Invitrogen PCR SuperMix HiFi

10uL Master Mix + 8.8uL associated H2O

O/n RNAi clone culture

1

T7 Sense primer 100mM stock

0.2 N.B: check pipetman - 0.2 may not register in tip; up to abt 0.8 if necc to see in tip.


2. Run on program RNAI-INS on PCR machine under MARK folder.



1 96C 3m

2 94 45s

3 58 45s

4 72 3m

5 goto 2 one time



6 94 45s

7 56 45s

8 72 3m

9 goto 6 one time



10 94 45s

11 54 45s

12 72 3m

13 goto 10 1 time



14 94 45s

15 52 45s

16 72 3m

17 goto 14 34 times



18 72 3m

19 10 forever



PCR SuperMix High Fidelity Kit Invitrogen P/N 10790-020

T7 Sense Primer IDT 5' CGT AAT ACG ACT CAC TAT AG 3'