Matthew Cai/LabNotes/2013-3-8
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First Strand cDNA synthesis[edit]
- Followed NEB E6300S protocol: [1]
Got 5ul aliquot of Human Brain Reference RNA from Rui at concentration 1ug/ul
- Used 1 ul for cDNA synthesis and put the rest in my box in -20C (Top of tube labelled HBBR)
- Mixed components in 0.2 ml microfuge tube labelled HBBR 1ug cDNA
Components | Volume |
RNA | 1 ul (1 ug) |
d(T)23VN (50 μM) | 2 ul |
H2O | 5 ul |
Total | 8 ul |
- Denatured RNA for 5 min at 70C and then put on ice
- Tightened the thermocycler too much and bent the tube slightly out of shape
- Added the following to tube
Components | Volume |
M-MuLV Reaction Mix | 10 ul |
M-MuLV Enzyme Mix | 2 ul |
Total | 20 ul |
- Incubated for one hour at 42C
- Inactivated enzyme for 5 min at 80C
- Stored at -20C for Second Strand synthesis on Saturday 3/9/13