PCR Laboration Companion,

Standardized for 4 RNA samples and 2 primer pairs

 

Reverse Transcription of mRNA for 5ml RT- and 25ml PCR Reaction Volumes

 

RT-Master mix for 28 tubes                                          

Oligo d(T)16                           0.25ml/tube            · 28 =       7 ml

RNA:ase inhibitor                0.25ml/tube            · 28 =       7 ml

10XPCR Buffer II                  0.5ml/tube              · 28 =       14 ml

25mM MgCl2(aq)                  1ml/tube                 · 28 =       28 ml

dNTP mix                               2ml/tube                 · 28 =       56 ml

 

MuLV-RT                              0.25ml/tube            · 28 =       7 ml

 

 

Make new mastermixes for 4 different RNA-templates, each prepared for 6.6 tubes

                In 4 separate empty eppendorf tubes: add RNA for 7 tubes       

0.75ml/tube            · 6.6 =      4.95 ml

 

Add RT-Master mix for 7 tubes into each of the 4 RNA-containing tubes

4.25ml/tube            · 6.6=       28.05 ml

(+ 4.95ml RNA=33 ml)

 

(Standard amount of RNA used is 25ng. GAPDH is amplified from 0.01ng RNA.)

(Add DEPC-H2O: if MuLV or RNA template have been omitted add DEPC-H2O to compensate)

 

You now have 4 Mastermixes with different RNA

                Distribute to the reaction tubes         5 ml per tube

 

 

 

Allow all tubes to incubate 10 minutes, allowing oligo d(T)16 extension by the RT-enzyme. Extended primers will remain annealed to the RNA template upon raising the reaction temperature to 42°C.

 

Perform RT in GeneAmp PCR 2400.

 

RT-reaction (approximate)

15 minutes at 42°C for reverse transcription

5 minutes at 99°C for denaturing

5 minutes at 5°C for cooling

PCR for two primerpairs and 25ml reaction volume

 

Master mix for 13 tubes                                    

Ampli-Taq Gold DNA-polymerase                    0.25ml/tube            · 13 =       3.25      

25mM MgCl2(aq)                                                  1ml/tube                 · 13 =       13          

10XPCR Buffer II                                                  2ml/tube                 · 13 =       26

dd H2O                                                                   16.25ml/tube          · 13 =       211.25

 

Primers

                Add upstream primer                                           0.25ml/tube            · 13 =       3.25      

                Add downstream primer                                     0.25ml/tube            · 13 =       3.25

 

Make two sets of the 13 tube Mastermix containing relevant primer pairs

Distribute to RT-tubes                                                        20ml/tube

 

Perform PCR cycling in GeneAmp PCR 2400

 

PCR-cycling (approximate)

12 minutes at 95°C for Ampli-Taq Gold activation, followed by a chosen number of cycles consisting of

15 seconds at 95°C for melting and

30 seconds at 60°C for annealing and extension. The last cycle terminates in

7 minutes at 72°C for final extension and

optional cold storage at 4°C.

               

 

Erik Edström, 20001127