Blue:RNA-Seq Experiments:06042014

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Revision as of 21:46, 3 June 2014 by >B1lake (Created page with " == Quartz-Seq Library Preparation == '''RT Primer Removal''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Purified cDNA||||||||||3ul |- |10x PCR Buffer||||||||||0.4...")
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Quartz-Seq Library Preparation

RT Primer Removal


Volume
Purified cDNA 3ul
10x PCR Buffer 0.4ul
10x Exo I buffer 0.2ul
100mM DTT 0.2ul
Exonuclease I 0.6ul
dH2O 1.6ul
Total 6ul
Takara Taq PCR Buffer:
Volume
1M Tris.HCl (pH 8.3) 100ul (100mM Final)
1M KCl 500ul (500mM Final)
1M MgCl2 15ul (15mM Final)
dH2O 385ul
Total 1ml


Add directly to beads and mix
Incubate @37C 30min
Incubate @80C 20min
Incubate @4C


Poly-A Tailing

Volume
ExoI RXN 6ul
10x PCR Buffer 0.5ul
10mM dATP 1.5ul
RNaseH 0.12ul
TdT enzyme (Roche 03333566001) 0.42ul
dH2O 2.46ul
Total 11ul


Incubate @37C 50 sec
Incubate @65C 10min
Incubate @4C


Second Strand Synthesis


Volume
Poly-A Tailed Sample 11ul
2x Terra Direct PCR Buffer 25ul
10uM Tagging Primer 0.32ul
Terra Polymerase 2ul
dH2O 18.68ul
Total 57ul
Incubate @98C 2min 10 sec
Incubate @40C 1min
Incubate @68C 5min
Transfer to ice


PCR Amplification

Volume
2nd Strand Sample 57ul
2x Terra Direct PCR Buffer 25ul
100uM PCR Primer 1ul
dH2O 24ul
Total 107ul


68C 1 sec
98C 10s -> 65C 15s -> 68C 5min 15x (1ng); 18x (100pg); 21x (10pg)
68C, 5min


Ran 5ul onto gel: