PCR protocols *


RT-PCR with a specific sequence primer *

First Strand Synthesis *

PCR reaction *





PCR protocols



10x PCR 3+ buffer:

(Maria R. Ponce and Jose L. Micol. NAR (1992), Vol. 20, No. 3, 623)

Tricine pH 8.4

300 mM


20 mM


30 mm






10x PCR buffer

10 ul

dNTPs 2 mM each

10 ul

DNA (1ng/ul)

5 ul

Forward primer (10 pmoles/ul)

5 ul

Reverse primer (10 pmoles/ul)

5 ul

Taq DNA polymerase (5u/ul)

0.5 ul



H2O to final reaction vol.

100 ul


RT-PCR with a specific sequence primer

Naz Islam 1996


5X First stand buffer [250 mM TRIS-HCl pH 8.3, 375 mM KCl and 15 mM MgCl2]

0.1 M DTT

10 mM dNTP mix (10 mM each dATP, dCTP, dGTP and dTTP)


10x PCR 3+ Buffer

First Strand Synthesis

Care must be taken to use only double (or single 45 min.) autoclaved solutions and pipettes.


1) poly A+ RNA (1 to 2g) or total RNA (5 g max.) x l

Primer (random primer 75ng, oligo-dT 500ng or degenerate primer 200ng) x l

H2O x l


Final reaction volume 20 l

2) Heat the mixture to 70oC for 10 min. and quick chill on ice. Collect the contents of the tubes by brief centrifugation and add the following:

5X First strand buffer

4.0 l

0.1M DTT

2.0 l

10 mM d(NTP) mix

1.0 l

[a32P]-dCTP (3000 Ci/mMol)




Final volume

18.0 ml

Mix the contents of the tube, briefly centrifuge and equilibriate the temperature by placing the vial at 37oC for 2 min.

3) Add 2.0 ml (200 U/ml) SUPERSCRIPT RT, mix gently and incubate at 37oC for 1h.

4) Place the tube on ice to terminate the reaction.

5) Remove 2 ml of the reaction and transfer to a microcentrifuge tube containing: 48 ml of 20 mM EDTA and 5 ml of yeast RNA (1 mg/ml)

6) To the remaining 18 ml add 6 ml 5M NaCl and 76 ml H2O. Extract once with 100 ml of phenol-chloroform 1:1 and once with chloroform alone.

7) Precipitate nucleic acid by adding 2.5 vol. EtOH, leave at -20oC for >1hr, spin 17 krpm, 20 min. (Sorvall SS34). Carefully remove S/N with pipette (keep in separate tube), add 200 ml 80% EtOH, -20oC, spin 10min 17 krpm and again carefully remove all S/N.

8) Dry pellet for 2 min. in SpeedVac and redissolve in 10 ml of H2O.

Analysis of cDNA products (first strand)



Dot 10 ml of the above mixture on DEAE-filter paper and transfer to a glass vial. For control, dot equivalent activity of [a-32P] dCTP and transfer to a separate glass vial. Repeatedly, wash the filter paper by adding fresh 0.5 M NaH2PO4 until no significant radioactivity remains on the control filter. Determine radioactivity and calculate yield by the following equation:

Amount of = (CPM) X(50 ml/ 10 ml) X (20 ml/2 ml) X (4 pmol dNTP/ pmol dCTP)

cDNA (mg) S.A (CPM / pmol dCTP) X (3,030 pmol dNTP / mg cDNA)

Specific activity (S.A) = CPM / 10 ml

200pmol dCTP / 10 ml


Gel analysis:

Precipitate DNA from remaining 40 ml of synthesis reaction withdrawn for analysis by adding 20 ml of NH4OAc and 2.5 vol. of EtOH. Wash the ppt. 1X with 70 % EtOH, dry and dissolve in 9 ml of TE. Add 1 ml of 1N NaOH. Apply sample on 1.4 % agarose gel after adding 2.0 ml of 5X loading buffer use end labelled l HIND III markers. Dry gel post electrophoresis and autoradiograph.

PCR reaction

10x PCR 3+ buffer

10.0 ml

2 mM d(NTP) mix

10.0 ml


10.0 ml

forward primer (50pM/ml)

2.0 ml (about 1 mg of a 25 mer)

reverse primer (50pM/ml)

2.0 ml (about 1 mg of a 25 mer)



Add H2O to Final volume

100.0 ml

Finally add 0.5 ml Taq DNA polymerase (5U/ml), mix carefully and spin, add 100ml mineral oil and begin PCR reaction:- 1min 94.5o C, then 35 cycles 30sec 94.5o C, 30sec 37o C, 30sec 72o C and finally 10 min at 72 o C.