RT PCR of Embryos

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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