Sam:LabNotes/Microbione/2009-2-5: Difference between revisions
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>Sam Chiang |
>Sam Chiang |
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Gel stain: 4uL SYBR safe (Invitrogen)/ 50 mL Agarose gel mix | Gel stain: 4uL SYBR safe (Invitrogen)/ 50 mL Agarose gel mix | ||
Small gel tray with 8-well comb | Small gel tray with 8-well comb | ||
Ladder: Low mass ladder (Invitrogen) | |||
Sample loading: (6 uL sample + 1 uL dye) -> load 6 uL mix | Sample loading: (6 uL sample + 1 uL dye) -> load 6 uL mix | ||
Loading order: L->R, #1~#8(02-04-09' PCR products) -> ladder -> #1~#6 (02-05-09' PCR products) | Loading order: L->R, #1~#8(02-04-09' PCR products) -> ladder -> #1~#6 (02-05-09' PCR products) | ||
Running at 135 V for 30 min. | Running at 135 V for 30 min. | ||
==Results== | ==Results== |
Revision as of 18:12, 8 February 2009
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
Step5: Agarose gel electrophoresis condition
1.5% agarose gel: 0.75 g + 50 mL (0.5 TBE). Heat up with 30 sec + 20 sec Gel stain: 4uL SYBR safe (Invitrogen)/ 50 mL Agarose gel mix Small gel tray with 8-well comb Ladder: Low mass ladder (Invitrogen) Sample loading: (6 uL sample + 1 uL dye) -> load 6 uL mix Loading order: L->R, #1~#8(02-04-09' PCR products) -> ladder -> #1~#6 (02-05-09' PCR products) Running at 135 V for 30 min.
Results
FIG. xxxxxxxxxxxxxxxxx
Discussion