Subject: Asilomar handout "Diff. Display" (R. Kahmann)

From: [email-masked] (Joerg Kaemper)
Date: 12 Apr 1995 11:52:11 -0700

Differential Display of eukaryotic mRNAs
(Liang and Pardee, Science 257, 967-971)

I. Reverse Transcription of mRNA

20 =B5L Final volume                      =B5L
dH2O                                    X
5X buffer                               4
DTT (0.1M)                              2
dNTP (250=B5M)                            1.6
mRNA                                    0.2 =B5g DNA-free total RNA
T11XY (10=B5M)                            2
______________________________
Total                                   19 =B5L

65=B0C, 5 min - 37=B0C, 70 min - 95=B0C, 5 min - 4=B0C
1 =B5L RTase (20 units) is added 10 min after at 37=B0C.
 At the end of RT spin the rube briefly to collect
 condensation. Set tubes on ice for PCR or store at -20=B0C
 for later use.

II. PCR

20 =B5L Final Volume                             =B5L
dH2O                                           9.2
10X PCR buffer                                  2
dNTP (25 =B5M)                                   1.6
35S-dATP (12.5mCi)                               1
L-primer (10=B5M) (10mer arbitrary primer 50%GC)  2
T11XY (10=B5M)                                    2
RT mix                                          2
Ampli Taq                                      0.2
____________________________
Total                                           20

Mix well by pipetting up and down. Add 25 =B5L mineral oil.
PCR: 94=B0C, 30 sec. - 40=B0C, 2 min - 72=B0C, 30 sec.
for 40 cycles - 72=B0C, 5 min - 4=B0C.
Use 3.5 =B5L of samples plus 2 =B5L of loading dye and incubate
 at 80=B0C for 2 min before loading onto a DNA sequencing gel (6%).

III. SECONDARY PCR:

1.      Cut out band from dried gel. Soak for 10 min in 100 =B5L of H2O.
2.      Boil for 10 min.
3.      Add:
        a.      1/10 vol NaOAc
        b.      50 =B5g glycogen (Boehringer)
        c.      300 =B5L of ethanol
4.      Incubate for 30 min at -80=B0C.
5.      Microfuge and wash with 80% ethanol.
6.      Dissolve in 10 =B5L of H2O.
7.      PCR (as for primary PCR), using:
        a.      5 =B5L of DNA
        b.      20 =B5M dNTPs
        c.      1 =B5M of each primer
        d.      40 =B5L final vol
8.      Run 20 =B5L on a gel and save the remainder for cloning or
reamplification.

-----   use In Vitrogen TA cloning kit or use fragments directly
to probe cDNA or genomic library

Joerg Kaemper
Institute of Genetics, University of Munich
Maria Ward Str. 1a, 80638 Munich, Germany
Phone: 49-89-17919821
=46ax: 49-89-17919820