Reagents for cDNA Synthesis and PCR found in -20^C Freezer in adjacent room.
All reagents must be kept on ice. Label One autoclaved PCR snap-cap tube per mRNA plus a negative control.
[NOTE: Make sure to keep a worksheet in notebook of cDNA and PCR reactions, primers added, et al.]
Bring Heating Block(s) (or Water Bath(s)) to 68^C and 37^C.
PREPARE cDNA SYNTHESIS REACTIONS BY COMPLETEING THE FOLLOWING:
* 1.0 microl Random Oligo (Diluted 1:8 = 0.5 mg/ml) [Boeringer Mannheim]
* Labelled as 1:8 Random Hex in 1.5 microl Ependorf Tube.
* 9.5 microl DEPC Water
* 10.0 microl 5X M-MLV RT Buffer [Gibco BRL]-Use at Full Strength. Labelled as First Strand Buffer.
* 10.0 microl POLY(A) + mRNA 2.0 microg/microl (=20microg) or DEPC Water Control
* 5.0 microl 100 mM DTT [Gibco BRL]
* 12.5 microl dNTPs at 2 mM each; STOCK IS 100 mM so dilute 1:50 [Eg: 4 microl G/4 microl A/4 microl T/4 microl C : 184 microl DEPC Water] [NOTE: Be sure to have enough dNTPs for PCR reaction]
* HEAT Reaction Tubes at 68^C for 10 minutes in 68^C Heating Block (or water bath).
* COOL Reaction Tubes to 37^C for 10 minutes in 37^C Heating Block (or water bath).
* ADD 1.0 microl RNasin at 40 U/microl [Promega] and 1.0 microl M-MLV Reverse Transcriptase at 200 U/microl [Promega]
* Heat at 37^C for 1 hour [NOTE: 50 ml volume should be total volume for each reaction tube]
Basic PCR protocol assumes 1.5 mM MgCl2. Procedure should be optimized for each particular primer pair.
Purchased PCR Kit (from University Stores) contains buffer and separate 25 mM MgCl2. [NOTE: Autoclaved 10X PCR Buffer may be prepared (100 mM Tris pH8.3, 500 mM KCl, 15mM MgCl2, 0.01% w/v gelatin)].
ADD REAGENT NOTES
* 66.5 microl ddH2O (64.5 ml ddH2O)
* 10.0 microl 10X PCR Buffer (10.0 microl 10X PCR Buffer [Promega or as above])
* 6.0 microl 25 mM MgCl2 (1.5 mM Final Concentration)
* 1.0 microl 250 ng/microl 5' Primer (5.0 microl 5' Primer at 5pM/microl (=25pM 5' Primer))
* 1.0 microl 250 ng/microl 3' Primer (5.0 microl 3' Primer at 5pM/microl (=25pM 3' Primer))
* 5.0 microl cDNA mix (From Synthesized cDNA from mRNA)
* 0.5 microl Taq (Enzyme) (At 4.5 U/ml [Promega])
* 100.0 microl Autoclaved PCR Oil (To Prevent evaporation during cycling)
PCR Machine Settings-25 Cycles
* 5 minute Initial-Time-Delay at 94^C [At 94^C Add 10 microl dNTPs at 2mM each]
* 1 minute 30 second Segment at 94^C
* 2 minute 30 second Segment at 54^C
* 3 minute 0 second Segment at 72^C
* Autoextension: YES 2.0 seconds per cycle (Attached to Segment #3)
* 7 minute incubation at 72^C
* Soak reaction tubes at 4^C overnight
Separate PCR Product from oil by either Bubbling through the oil with the pipette and aspirating the aqueous solution beneath or freeze the Reaction tube(s) overnight at -80^C. Thaw briefly and aspirate oil off while aqueous layer is still frozen. Check PCR product by running on a 0.5-2% agarose gel.
Protocol submitted by:
by David Richard Nelson
from: [Molecular Cloning: A Laboratory Manual; Second Edition. J. Sambrook, E.F. Fritsch, T.Maniatis. Cold Spring Harbor Laboratory Press, ©1989.]