RT PCR of Embryos
RT
RNA: 10 embryos snap frozen at various stages in liquid N2
Prepare using RNeasy columns, elute in 50 uL RNase free DI H2O
20 uL RT Reactions:
5 uL RNA ) 13 uL total can vary amounts
8 uL H2O )
1 uL oligo dT (1.5 mg / mL)
4 uL 5 X RT Buffer
0.33 uL BSA (3 mg / mL)
0.5 uL dNTPs (25 mM stock)
0.5 uL RNasin
0.5 uL MMLV-RT
Combine RNA, H2O, and Oligo-dT. Heat to 65C for 3 minutes.
Cool on ice, spin down briefly.
Add mix with remainder of ingredients.
Add 6 uL to each reaction.
Incubate at 42C for 30 min to 1 hour
(To remove contaminating DNA before starting, treat with DNAse and repeat RNeasy column to cleanup)
10 uL PCR Reactions:
4 uL RT Reaction
1 uL 10 X PCR buffer
0.1 uL dNTPs (25 mM stock, add 1:50 32P-nucleotide)
0.5 uL up Primer (1 mg / mL)
0.5 uL down Primer (1 mg / mL)
3.8 uL H2O
0.1 uL Taq
(32P-CTP = 0.2 uL)
PCR machine steps:
94C 3 min
94C 1 min**
55C 1 min**
72C 1 min**
72C 5 min
4C Hold
** Repeat 15 – 25 X depending on template