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

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>Sam Chiang
(New page: ='''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 T...)
 
>Sam Chiang
(Removing all content from page)
 
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='''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 (10 uL 100 uM Forward + 10 uL 100 uM Reverse + 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'''
[[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 20:51, 16 March 2009