2 Enzymatic Modification of DNA and RNA
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Materials
• Thermal cycler programmed to 37°C/60 min, 65°C/5 min, 4°C/hold for
reverse transcription. For the subsequent PCR amplification, choose optimal cycling conditions according to the principles in chapter 12.
• total cellular RNA (0.5-1 f.lg/f.ll) in DEPC-treated water.lt is best to check
the integrity of the RNA preparation in a formaldehyde agarose gel.
• Primer: There are three options to prime first strand synthesis.
- (dT) 12_18 (0.5 f.lg/f.ll in water) which hybridizes to the 3' poly(A) tail of
mRNA molecules
- Random hexamers, which hybridize to all RN A molecules presen t and
are used when a particular mRNA molecule is difficult to copy in its
entirety, 50 ng/f.ll in water
- Specific antisense primers which hybridize only to the target mRNA
(10 pmol/f.ll in water; for 21-mer, this is approximately 70 ng/f.ll)
• lOx buffer, 200 mM Tris-HCl, pH8.4, 500 mM KCl, 25 mM MgCh, 1 mg/ml
bovine serum albumine
• DTT, 0.1 M
• dNTP mix, 10 mM each dATP, dCTP, dGTP, dTTP in water
• RNase inhibitor (RNasin®, Promega)
• MMLV reverse transcriptase, 200 U/f.ll. Use approximately 200 U/f.lg
RNA
• PCR primers 1 (forward) and 2 (reverse), each at 20 pmol/f.ll
• Taq DNA polymerase
• DEPC-treated water
Procedure
1. On ice, pipet the following into a 0.5 ml microcentriguge tube:
Total RNA
Prim er
2. Heat to 70°C for 10 min.
lJ.lg in DEPC treated water
In the above concentration
Supplies
Reagents
and solutions
MMLV reverse
transcriptase