Sam:LabNotes/Microbiome/2009-2-26: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Sam Chiang
(New page: ='''Testing of putative amplification from human cell lysate MDA test 2 (on 02-25-09)'''= ==Objective== *Using regular PCR (Taq enzyme 2X) to confirm 8 putative amplicons. ==Material...)
 
>Sam Chiang
(Removing all content from page)
 
Line 1: Line 1:
='''Testing of putative amplification from human cell lysate MDA test 2 (on 02-25-09)'''=


==Objective==
*Using regular PCR (Taq enzyme 2X) to confirm 8 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
**18S-306 (cho. 21) - previously tested
**2-1 (cho.2, 540 bp, Tm 52C)
**3-1 (cho.3, 235 bp, Tm 52C)
*Templates (1 uL): A3, A6, A7, B4, B7, C3, C4, C8, D6(p-Pos), D7(p-Neg), Pos(1/100 gDNA = 1 ng/uL), H2O
==Exp. Design==
         
                                            Templates (A->H)
            ---------------------------------------------------------------------------
                                A3  A6  A7  B4  B7  C3  C4  C8  p-Pos  p-Neg  Pos  H2O
            ---------------------------------------------------------------------------
  Primer  Strip1 P1 (18S-306)   
            stirp2 P2 (2-1)   
            strip3 P3 (3-1)
==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      14 rxn
      ----------------------
      H2O    3.5      49.0
      Primer  0.5        7.0  - primer is differnt in each one of three master mix
      Taq 2X  5.0      70.0
      ----------------------
              9.0      126.0 (uL)  126/14=9 
*Transfer 9 uL of master mix into each reaction
*Perform PCR reaction
**Strip 1 goes to block A (program GENE59: Tm59, 30 cycles)
**Strip 2-3 go to block B (program GENE52: Tm52, 30 cycles)
*Gel Electrophoresis
**1.5% agarose: (1.5 g agarose +100 mL 0.5X TBE + 8 uL SYBR safe -> four small tray with 8-well comb)
**Run at 135 V for 20 min.
==Results==
'''FIG. Gel electrophoresis picture '''
  [[Image:ZhangLab_2 2009-02-26 test2 primer_18S.bmp|600px]] [[Image:low mass ladder.bmp|150px]]
  '''Test by primer 18S (306 bp)'''
  [[Image:ZhangLab_2 2009-02-26 test2 primer_2-1.bmp|600px]]
  '''Test by primer 2-1 (540 bp)'''
  [[Image:ZhangLab_2 2009-02-26 test2 primer_3-1.bmp|400px]]
  '''Test by primer 3-1 (235 bp)'''
==Result & Discussion==
*Samle B7 and p-Pos contains positive bands by 18S primer (Cho.21/Un) and primer 3-1 (Cho.3).
*A very weak band was also found (but not showing clearly in pic) on B7 by primer 2-1 (Cho.2). However the p-Pos failed to show it.
**Sample p-Pos should contain successful amplification. The faliure of p-Pos may be caused by the interference of junk (salt?) left in MDA PCR product.
**Clean the junk using PCR product purification kit and test the PCR again on those p-Pos sample.

Latest revision as of 21:05, 16 March 2009