Search

PCR Primer Design and Reaction Optimisation

Reverse Transcription PCR

RNA -> LOTS OF DNA

(modified from PCR Protocols January 1991 )

Ed Rybicki

copyright 1992, 1996


Contents

Reverse Transcription Reaction

Polymerase Chain Reaction

 


Reverse Transcription Reaction:

This provides the cDNA - by extension from a primer complementary to the RNA sequence - for the amplification by PCR using 2 primers.

REMEMBER TO USE A PRIMER WHOSE SEQUENCE IS COMPLEMENTARY TO THE RNA!!

Reverse Transcription (RTase) Reaction Mix:

It is convenient to make up a single master mix to be aliquotted out for a number of RNAs to be reverse transcribed using the same primer.

sample mix: per 10ul

  • 2ul RNA preparation
  • 1 ul DMSO
  • 1 ul 40 U/ul RNAsin
  • 6ul DEPC-H20 per reaction:

NB: master rxn mix made separately!!

Total volume master rxn mix:

  • 22ul X N (no. samples) + 5% 1/10 total vol
  • 1 OmM dNTPs
  • 1/5 vol BRL 5x RTase buffer [MuLV enzyme]
  • 1/20 vol 20 U/ul MMuLV RTase [NB: dilution of 200U/ul stock ! !]
  • 0.5 ug specific primer [eg. 0.3ul of 288uM=1.9ug/ul 20-mer]
  • 1/20 vol DMSO [dimethyl sulphoxide]
  • DEPC-H20 to 22ul/rxn:

REMEMBER TO ALLOW FOR 10ul/rxn FOR SAMPLE!!

Mix everything together, leave on ice

HEAT SAMPLE MIX AT 65 C FOR 3 MIN, --> WET ICE

ADD 10ul RTase MIX / SAMPLE

MIX BY VORTEXING, HEAT SAMPLES AT 42 C 60 min / 52 C 30 min

 


Polymerase Chain Reaction (modified from PCR Protocols)

Reaction Mix:

It is again convenient to make up a master mix to be aliquotted out to amplify all samples, if they are to use the same primers. If not, modify master mix by simply leaving out primers.

Total volume: 50ul / rxn x N + 5-10% for wastage

Reaction master mix:

  • 1/10 vol Cetus/ Promega /other 1 0x buffer
  • 1/50 - 1/25 vol 2.5mM stock dNTPs [--> 50-100uM]
  • 1/20 vol 10uM forward (=RNA sense) primer [--> 0.5uM]

[NB: no reverse primer needed IF THIS IS THE.SAME AS cDNA PRIMER as

residual cDNA synthesis primer concn. is +/- 5uM]

  • OPTIONAL: 1/20 - 1/10 vol DMSO
  • 0.5ul of 5U/ul Taq polymerase / 100ul rxn mix

REMEMBER TO ALLOW FOR 5ul/rxn OF SAMPLE!!

  • MIX MASTER RXN MIX, LEAVE ON ICE.
  • Aliquot out 45ul / PCR rxn vial
  • Add 5ul sample / vial from reverse transcription reaction mix
  • Add 50ul / vial mineral oil (NB: new upipette tips each time!!)
  • Vortex lightly, spin down

PCR Conditions: recommended:

  • 94o C 3 min; 45-50oC 3 min; 72oC 3 min;
  • (93oC 1 min; 45-50oC 1 min; 72oC 1-3 min) x 30-34 cycles
  • 72oC 5-10 min