Sam:LabNotes/Microbione/2009-2-5: Difference between revisions
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='''Testing human 18S using regular PCR'''= | ='''Testing human 18S using regular PCR'''= | ||
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#Using different conc. of human gDNA to optimize the condition. | #Using different conc. of human gDNA to optimize the condition. | ||
#Using Taq 2X master mix (New England Biolabs) enzyme which is newly opened. | #Using Taq 2X master mix (New England Biolabs) enzyme which is newly opened. | ||
==Reagents== | ==Reagents== | ||
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#Enzyme - Taq 2X master mix (New England Biolabs) | #Enzyme - Taq 2X master mix (New England Biolabs) | ||
#Template - diluted human genome DNA (Jurkat cell, New England Biolabs | #Template - diluted human genome DNA (Jurkat cell, New England Biolabs | ||
#18S primer mix (10 uM | #18S primer mix (10 uM Forward + 10 uM Reverse) -> hS18_212, hS18_306 (10 uL 100 uM Forward + 10 uL 100 uM Reverse + 80 uL H2O) | ||
==Exp. Design== | ==Exp. Design== | ||
Template | Template gDNA (100 ng/uL) H2O | ||
------------------------------ | |||
1/10 1/100 1/1000 Dilution factor | |||
------------------------------ | |||
Primer: 18S-211 #1 #2 #3 #4 (#1, #2 - replicates) | Primer: 18S-211 bp #1 #2 #3 #4 (#1, #2 - replicates) | ||
18S-306 #5 #6 #7 #8 (#4, #5 - replicates) | 18S-306 bp #5 #6 #7 #8 (#4, #5 - replicates) | ||
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Machine: Bio-Rad 2 blocks- DNA engine | Machine: Bio-Rad 2 blocks- DNA engine | ||
Program name: Gene59 | Program name: Gene59 | ||
94C 2min -> (94C 40 sec -> 59C 40 sec -> 72C 1 min ) x 29 cycle -> 72C 10 min -> 4C Forever | 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 (02-05-09' PCR products) -> #1~#6(02-04-09' PCR products) -> ladder | |||
Running at 135 V for 30 min. | |||
==Results== | |||
'''FIG. Gel picture of 18S primers testing''' | |||
[[Image:ZhangLab_Gel pic 020609-new.jpg|600px|none]] | |||
#02-04-09' results: PCR products #1 and #2 - duplicates (primer S18_211, amplicon 211 bp) , #4 and #5 - duplicates (primer S18_306, amplicon 306 bp), #3 and #6 blank (no template control) | |||
#02-05-09' results: PCR products #1, #2 and #3 - template (10 ng, 1 ng, 100 pg respectively. Primer S18_211, amplicon 211 bp) , #4 - blank; #5, #6 and #7 - template (10 ng, 1 ng, 100 pg respectively. Primer S18_306, amplicon 306 bp) , #8 - blank | |||
==Discussion== | |||
#The band specificy is acceptible for both S18 primers, and no contaminatino in reagents was found. | |||
#Both condition of both realtime PCR and coventional PCR were working and appliable for following experiments. Both EconoTaq 2X Master and NEB Taq 2X Master work well in these amiplifications. | |||
#The results from 02-04-09' indicated that realtime PCR reaction was acturally successful for 20-cycle amplification. It's unknow why the real-time monitoring of signal curve was shown correctly on the Chromo4 machine. (The SYBR 2X didn't fucntion?) | |||
#The results from 02-05-09' indicated that the dilution of template is effective to be seen on agarose gel. | |||
==Suggestion== | |||
#prepared a new tube of SYBR-2X and test it with old SYBR-2X regent using a small-scale realtime PCR reaction. |
Latest revision as of 23:27, 23 February 2009
Testing human 18S using regular PCR[edit]
Objective[edit]
- 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[edit]
- 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 (10 uL 100 uM Forward + 10 uL 100 uM Reverse + 80 uL H2O)
Exp. Design[edit]
Template gDNA (100 ng/uL) H2O ------------------------------ 1/10 1/100 1/1000 Dilution factor ------------------------------ Primer: 18S-211 bp #1 #2 #3 #4 (#1, #2 - replicates) 18S-306 bp #5 #6 #7 #8 (#4, #5 - replicates)
Preparation[edit]
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[edit]
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 (02-05-09' PCR products) -> #1~#6(02-04-09' PCR products) -> ladder Running at 135 V for 30 min.
Results[edit]
FIG. Gel picture of 18S primers testing
- 02-04-09' results: PCR products #1 and #2 - duplicates (primer S18_211, amplicon 211 bp) , #4 and #5 - duplicates (primer S18_306, amplicon 306 bp), #3 and #6 blank (no template control)
- 02-05-09' results: PCR products #1, #2 and #3 - template (10 ng, 1 ng, 100 pg respectively. Primer S18_211, amplicon 211 bp) , #4 - blank; #5, #6 and #7 - template (10 ng, 1 ng, 100 pg respectively. Primer S18_306, amplicon 306 bp) , #8 - blank
Discussion[edit]
- The band specificy is acceptible for both S18 primers, and no contaminatino in reagents was found.
- Both condition of both realtime PCR and coventional PCR were working and appliable for following experiments. Both EconoTaq 2X Master and NEB Taq 2X Master work well in these amiplifications.
- The results from 02-04-09' indicated that realtime PCR reaction was acturally successful for 20-cycle amplification. It's unknow why the real-time monitoring of signal curve was shown correctly on the Chromo4 machine. (The SYBR 2X didn't fucntion?)
- The results from 02-05-09' indicated that the dilution of template is effective to be seen on agarose gel.
Suggestion[edit]
- prepared a new tube of SYBR-2X and test it with old SYBR-2X regent using a small-scale realtime PCR reaction.