Sam:LabNotes/Microbiome/2009-2-25/exp1: 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
(Removing all content from page)
 
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='''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==
*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)
*Gel Electrophoresis
**1.5% agarose: (1.5 g agarose +100 mL 0.5X TBE + 8 uL SYBR safe)
**Run at 135 V for 30 min.
==Results==
'''FIG. Gel electrophoresis picture '''
  [[Image:ZhangLab_2 2009-02-26 09hr 46min copy.bmp|600px]] [[Image:low mass ladder.bmp|150px]]
  '''Left gel: test by primer 2-1 (540 bp); Right gel: test by primer 3-1 (235 bp)'''
  [[Image:ZhangLab_2 2009-02-26 12hr 04min.bmp|600px]]
  '''Left gel: test by primer 4-1 (383 bp); Right gel: test by primer 18S (306 bp)'''
==Discussion==
*None of four putative polonies has contained the human genomic DNA
*The n-Neg (Negitive control in MDA) has no band, which means the contamination is not human genome either.
*The 10 uL reaction volume works fine for this applification (PCR testing) and is able to save more enzyme for multiple reaction.
*The ladder is not shown well on 100, 200, 300 bp band even 6 uL of ladder was used.
==Suggestion==
*For gel electrophoresis, running at 135V, 20 min is sufficient.
 
*Need to repeat the MDA using different scale of dilution method and try to decrease the background in MDA reaction.
*It is also possible the salts left in MDA reaction inhibited the following ragular PCR reaction (PCR products need to be purified before they can serves as new templates).

Latest revision as of 21:03, 16 March 2009