Sam:LabNotes/Microbione/2009-2-25: Difference between revisions

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>Sam Chiang
(New page: ='''Testing of putative amplification from human cell lysate MDA on 02-24-09'''= ==Objective== *Using regular PCR (Taq enzyme 2X) to confirm four putative amplicons. ==Materials== *...)
 
>Sam Chiang
Line 1: Line 1:
='''Testing of putative amplification from human cell lysate MDA on 02-24-09'''=
*'''[[Testing of putative amplification from human cell lysate MDA on 02-24-09]]'''


 
*'''[[Human cell lysats MDA - test 2]]'''
 
==Objective==
*Using regular PCR (Taq enzyme 2X) to confirm four putative amplicons.
 
 
==Materials==
 
*Enzyme - Taq 2X master mix (New England Biolabs)
*Positive control template - 1/100 diluted Jurkat cell genomic DNA (NEB) -> 1 ng/uL
*Human genome primers
**1-1 (cho.1, 270 bp, Tm 52C)
**2-1 (cho.2, 540 bp, Tm 52C)
**3-1 (cho.3, 235 bp, Tm 52C)
**4-1 (cho.4, 383 bp, Tm 52C)
**18S-306 (cho. 21) - previously tested
 
*Templates (1 uL): B2, B4, B5, C3, E3(p-Neg), Pos(1/100 gDNA), H2O
 
 
 
==Exp. Design==
         
                                            Templates (A->H)
            -------------------------------------------------------
                                    B2  B4  B5  C4  p-Neg Pos H2O
            -------------------------------------------------------
  Primer  Strip1 P1 (1-1)   
            stirp2 P2 (2-1)   
            strip3 P3 (3-1)
            strip4 P4 (4-1)
            strip5 P5 (18S-306)
 
 
==Procedures==
*Thaw the Taq2X enzyme and primers.
 
*Set up 52C program.
 
*Preppare 5 trips of 8-well PCR tubes and transfer the 1 uL template.
 
*Master Mix - x 5 tubes:
            1 rxn      8 rxn
      ----------------------
      H2O    3.5      28.0
      Primer  0.5        4.0  - primer is differnt in each one of five master mix
      Taq 2X  5.0      40.0
      ----------------------
              9.0      72.0 (uL)  72/8=9 
 
*Transfer 9 uL of master mix into each reaction
 
*Perform PCR reaction
**Strip 1-4 go to block A (program GENE52: Tm52, 30 cycles)
**Strip 5 goes to block B (program GENE59: Tm59, 30 cycles)

Revision as of 19:02, 26 February 2009