Editing
Sam:LabNotes/Microbione/2009-2-4
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
=Testing human 18S using realtime PCR= ==Objective== #Prepare the primers (human 18S and Bacteria 16S) for the future use in MDA experiments as positive control confirmation (The Bac 16S primer is based on given sequecne from Dr. Pillai and Dr. Dowd's lab. The blat results showed these sequence match most of E. coli genome). Primers info are referred to [[Sam:LabNotes/Microbione/2009-2-3]] #Practice realtime PCR machine operation #Optimaize primer and template concentration and Tm using EconoTaq 2X master enzyme ==Reagents== #H2O - (Ambion, bottled Nuclease-free water) leave in PCR hood for UV decontamination #Dye - 2X SYBR (working sol.)- diluted from 10,000X (Stock sol.)-> 50X (Stock sol.), only 50X and 2X (diluted) SYBR can be kept in 4 degree #Enzyme - EconoTaq 2X master - kit #Template - (10 ng/uL)human genome DNA from Jurkat (Human immortalized T-cell line) #Primer mix (10 uM forward + 10 uM Reverse) ==Exp. Design== Template gDNA H2O -------- --- Primer: 18S-211 #1 #2 #3 (#1, #2 - replicates) 18S-306 #4 #5 #6 (#4, #5 - replicates) ==Preparation== '''Primer diluaiton''': Oligo tube -> add H2O (10 times nmole) uL -> 100 uM 10 uL of 100 uM (forward) + 10 uL of 100 uM (reverse) + 80 uL H2O => Primer mix '''gDNA Template dilution''': Stock solution 100 ug/mL = 100 ng/uL Oligo tube 5 uL of 100 ng/uL + 45 uL H2O -> 50 uL of 10 ng/uL ==Procedures== Step1: Preparing master mix 1 (6+1) rxn -------------------------------------------- H2O 7.0 49.0 uL SYBR(2X) 1.0 7.0 uL EconoTaq 2X Master 10.0 70.0 uL -------------------------------------------- 18.0 126.0 uL(126/7=18) Step2: Transfer 18 uL of Master mix into each of 6 individual PCR tubes Step3: Add 1 uL primer (h18S-211 or h18S-306) and 1 uL template (gDNA or H2O) as indicated in Exp. design Step4: Perform real-time PCR Program name: sam020409 94C 2min -> (94C 45 sec -> 58C 2 min -> 72C 1 min -> Plate read) x 20 clcle -> 72C 5 min -> 15C hold ==Results== '''FIG.1 Realtime amplification plot (no log in flourescent)''' [[Image:sam020409.jpg|600px|none]] '''FIG.2 Realtime amplification plot (with log in flourescent)''' [[Image:sam020409-log.jpg|600px|none]] ==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)
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information