Sam:LabNotes/Microbione/2009-2-5: Difference between revisions
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=Follow up: Testing human 18S primers using realtime PCR= | ='''Follow up: Testing human 18S primers using realtime PCR'''= | ||
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=Testing human 18S using regular PCR= | ='''Testing human 18S using regular PCR'''= | ||
Revision as of 16:59, 6 February 2009
Follow up: Testing human 18S primers using realtime PCR
Exp. Design
Template gDNA H2O -------- --- Primer: 18S-211 #1 #2 #3 (#1, #2 - replicates) 18S-306 #4 #5 #6 (#4, #5 - replicates)
Results
FIG.1 Realtime amplification plot (no log in flourescent)
FIG.2 Realtime amplification plot (with log in flourescent)
Discussion
- The amplification curves are not distinguishable (Sample vs. Blank) within 20 clycles.
- The number of amplifications cycles we used is not enough?
- The amplifications are not working? (Primers' specificity? Concentration of templates?)
Suggestion
- Perform the Gel electrophoresis for the amplicons -> check the primer specificity
- Repeat the same reactions using regular PCR and bigger cycles(without SYBR)
Testing human 18S using regular PCR
Objective
- Check if the 18S primers are specific by agarose gel.
- Using different conc. of human gDNA to optimize the condition.
- Using Taq 2X master mix (New England Biolabs) enzyme which is newly opened.
Reagents
- H2O - (Ambion)
- Enzyme - Taq 2X master mix (New England Biolabs)
- Template - diluted human genome DNA (Jurkat cell, New England Biolabs
- 18S primer mix (10 uM forward + 10 uM Reverse) -> hS18_212, hS18_306
Exp. Design
Template gDNA (100 ng/uL) H2O ------------------------------ 1/10 1/100 1/1000 Dilution factor ------------------------------ Primer: 18S-211 #1 #2 #3 #4 (#1, #2 - replicates) 18S-306 #5 #6 #7 #8 (#4, #5 - replicates)
Preparation
gDNA Template dilution: Stock solution 100 ug/mL = 100 ng/uL Oligo tube Dilution from 100 ng/uL ->10 ng/uL (1/10), 1 ng/uL (1/100), 100 pg/uL (1/1000)
Procedures
Step1: Preparing master mix
1 8+2 rxn -------------------------------------------- H2O 8.0 80.0 uL Taq 2X Master Mix 10.0 100.0 uL -------------------------------------------- 18.0 180.0 uL(180/10=18)
Step2: Transfer 18 uL of Master mix into each of 8 wells in PCR tube-strip
Step3: Add 1 uL primer (h18S-211 or h18S-306) and 1 uL template (gDNA or H2O) as indicated in Exp. design
Step4: Perform regular PCR
Machine: Bio-Rad 2 blocks- DNA engine Program name: Gene59 94C 2min -> (94C 40 sec -> 59C 40 sec -> 72C 1 min ) x 29 cycle -> 72C 10 min -> 4C Forever