Sam:LabNotes/Microbione/2009-2-25/
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Testing of putative amplification from human cell lysate MDA on 02-24-09[edit]
Objective[edit]
- Using regular PCR (Taq enzyme 2X) to confirm four putative amplicons.
Materials[edit]
- 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[edit]
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[edit]
- 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[edit]
FIG. Gel electrophoresis picture
File:ZhangLab 2 2009-02-26 09hr 46min copy.bmp File:Low mass ladder.bmp Left gel: test by primer 2-1 (540 bp); Right gel: test by primer 3-1 (235 bp)
File:ZhangLab 2 2009-02-26 12hr 04min.bmp Left gel: test by primer 4-1 (383 bp); Right gel: test by primer 18S (306 bp)
Discussion[edit]
- 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[edit]
- 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).