Needed to synthesize more cDNA from 1ug total RNA to run 2 sets of probe capture reactions in parallel due to 2 types of primers and not knowing which will work better
First Strand cDNA synthesis[edit]
- Followed NEB E6300S protocol: [1]
From the 4ul of Human Brain Reference RNA 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
Second Strand cDNA synthesis[edit]
- Follow NEB E6111S protocol: [2]
- Added 48 ul H20
- Added 8ul 10x Second strand synthesis reaction buffer
- Added 4ul Second strand synthesis enzyme mix
- Mixed by pipetting
- Incubated for 2.5 hours at 16C
- Purified using Qiagen minelute column (eluted 23ul into 1.5ml tube labelled HBRR cDNA 3.11.13)
- Measured DNA conc with Nandrop: 36.1 ng/ul
- 21.5ul (0.776 ug) sample left in tube
cDNA + RNase digestion[edit]
- Did 2 more first strand and second strand cDNA reactions like above
- Stopped before purification to digest RNA and then purify tomorrow
- Stored samples in -20C freezer