Bioprotocols

Normal 0 7.8 pt 0 2 false false false MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:#0400; mso-fareast-language:#0400; mso-bidi-language:#0400;}

PROTOCOL FOR SINGLE CELL mRNA AMPLIFICATION (REVERSE NORTHERN ANALYSIS)

source: dartmouth.edu

I. First round amplification

A.First strand cDNA synthesis

1.Add to single cell:

             

Cell contents plus recording solution                    3ul

1x RT buffer                             17.5ul

dGTP (10mM)                               1ul

dATP (10mM)                                        1ul

dTTP (10mM)                                        1ul

dCTP (10mM)                                        1ul

Oligo-dT-T7(100ng/ul)                          1ul

DTT(100mM)                                         3ul

RNasin                                  0.5ul

AMV-RT(25U/ul)                             1ul

                                    

 

total volume                                  30ul

 

2.Mix gently; incubate at 42C for 90min.

3.Phenol/chloroform extraction cDNA:

 

              DD-H2O                                 105ul

              3M Na acetate                                       15ul

              chloroform                                     75ul

              buffer-saturated phenol                        75ul

 

              final volume                                       300ul

 

4.Vortex for 10 sec. Centrifuge for 3 min. Carefully remove~145ul aqueous (top)

       phase to a new tube.

5.Precipitate with ethanol. Add 300ul 100% ice-cold ethanol,1ul tRNA(5ug)

6.Leave on dry ice for least 20-30 mins. . Centrifuge at 18,000rpm at 4C for 30 min.

 

B.Second strand cDNA synthesis

 

7. Dry & resuspend pellet in 20ul DDH2O.

8.Heat at 90-95C for 3 min to denature RNA:DNA hybrid.

9. Cool quickly on ice; centrifuge briefly to bring down condensation.

10.Add to sample:                                              (20ul)

 

       10x 2nd strand buffer                                   5ul

       100mMDTT                                           2ul

       4 dNTPs(2.5mM each)                                  5ul

       random hexamers (100ng/ul)                               1ul

        T4 DNA polymerase (5U/ul)                            0.2ul(1U final)

       Klenow (5U/ul)                                    0.5ul(2U final)

       DDH2O                                       16.3ul

 

       final volume                                          50ul

11.Mix gently; incubate at 14C for a minimum of 5 hrs, or overnight.

 

C.Blunt-end treatment