First Strand cDNA synthesis
Dr. Stacey Smith is the "I" in this protocol
Objective: to make complementary (cDNA) from total RNA. Note that if you are planning to make cDNA for QPCR or RT-PCR, the RNA should have already been treated with DNase.
In a 0.5ml tube, combine on ice:
1.0 ul of 10mM dNTPs
2.0 ul of 10um Oligo dT-GR *
x.x ul of total RNA (0.2-2.0 ug, I recommend 0.5 to 1ug)
x.x ul of water (sterile, RNase free)
12.0 ul total
Using the old BOZO PCR machines, incubate the tubes at 65 C for 5min (to make all single stranded) then return to the ice.
To each tube, on ice, add:
4.0ul First Strand Buffer
2.0ul 0.1 mM DTT
1.0ul RNase OUT
19.0 ul (adding up from beginning)
Spin down quickly in small cfg to collect contents. Incubate on the BOZO at 37 C for 2 min. Keeping on the 37 C block, add:
1.0ul MMLV RT**
Mix well by pipetting. Incubate for an additional 50 minutes at 37 C to do the synthesis reaction.
Kill the reaction by incubating at 70 C for 15minutes.
You cannot quantify this cDNA with the spec or the nanodrop because there is RNA, dNTPS and primer in there, but assuming the reaction proceeded well, you should have converted all the RNA into cDNA. So if you put 0.5 ug of total RNA into the reaction, you should have 0.5ug of cDNA afterwards (for 20ul, that will be 25ng/ul).
*You could also use plain oligo dT, but using the GR version adds an adaptor that allows you to do 3'RACE with the GR-3' primer. And it can't hurt to use it even if you won't do 3'RACE.
**We use MMLV reverse transcriptase but there are other RTs that vary in their efficiency, etc. The important thing, if you are planning to use these cDNAs for quantification and comparison across tissues or individuals, is to be consistent and always use the same RT and cDNA synthesis conditions.