Sam:LabNotes/Microbione/2009-2-5: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Sam Chiang
No edit summary
>Sam Chiang
Line 13: Line 13:
#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 forward + 10 uM Reverse) -> hS18_212, hS18_306  
#18S primer mix (10 uM Forward + 10 uM Reverse) -> hS18_212, hS18_306
** 10 uL 100 uM Forward + 10 uL 100 uM Referse + 80 uL H2O


==Exp. Design==
==Exp. Design==
  Template                gDNA (100 ng/uL)    H2O
  Template                gDNA (100 ng/uL)    H2O

Revision as of 23:26, 23 February 2009

Testing human 18S using regular PCR

Objective

  1. Check if the 18S primers are specific by agarose gel.
  2. Using different conc. of human gDNA to optimize the condition.
  3. Using Taq 2X master mix (New England Biolabs) enzyme which is newly opened.


Reagents

  1. H2O - (Ambion)
  2. Enzyme - Taq 2X master mix (New England Biolabs)
  3. Template - diluted human genome DNA (Jurkat cell, New England Biolabs
  4. 18S primer mix (10 uM Forward + 10 uM Reverse) -> hS18_212, hS18_306
    • 10 uL 100 uM Forward + 10 uL 100 uM Referse + 80 uL H2O

Exp. Design

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

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 (02-05-09' 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

  1. 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)
  2. 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

  1. The band specificy is acceptible for both S18 primers, and no contaminatino in reagents was found.
  2. 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.
  3. 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?)
  4. The results from 02-05-09' indicated that the dilution of template is effective to be seen on agarose gel.


Suggestion

  1. prepared a new tube of SYBR-2X and test it with old SYBR-2X regent using a small-scale realtime PCR reaction.