Editing
Sam:LabNotes/Microbione/2009-3-3
(section)
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!
='''Human genome screening primer set - quality validation'''= ==Objective== *Human whole genome primer sets were obtained from Professor (2 sets). *The primer set were used to confirm the existence of chromosomes in MDA amplicon using multiplex PCR. *Individual validation of each primer is required before performing multiplex PCR. ==Samples & Materials== *Enzyme - Taq 2X master mix (NEB) *Templates (1 uL): 1 ng/uL human gDNA (NEB) *Primers: All primers are diluted to 100 uM, and mixed basd on the gene (Forward 20 uL + Reverse 20 uL + H2O 160 uL) **Set1: 1-1 ~ 22-1 and X-1, Y-1 (24 rxns) **Set2: 1-2 ~ 22-2 and X-2 (23 rxns) ==Procedures== *Thaw the Taq2X enzyme and primers. *Dilute all MDA template. Prepare diluted Pos template (1 ng/uL) *Set up PCR program (GENE59, GENE52). *Master Mix - x 2 (Primers are various) **Total reactions: 12 rxns 1 rxn 24+3 rxn --------------------------- H2O 3.5 94.5 Template 1.0 27.0 Taq 2X 5.0 135.0 --------------------------- 9.5 256.5 (uL) 256.5/27=9.5 *Transfer 9.5 uL of master mix into each of 24 PCR tubes (8-well strip) *Transfer the 0.5 uL primer into each reaction *Perform PCR reaction in thermocycler: program GENE52 (Tm52, 30 cycles) *Gel Electrophoresis **1.5% agarose: (1.5 g agarose +100 mL 0.5X TBE + 8 uL SYBR safe -> 2 medium tray with 26-well comb **Run at 135 V for 18 min. **Loading: Sample 7 uL + 2 uL loading buffer -> 7 uL. Diluted ladder 7 uL *Repeat the same design to perform PCR on primer set2 (23 reactions) ==Results== [[Image:ZhangLab_2 2009-03-03 15hr 01min-set1-new.jpg|600px]] [[Image:ZhangLab_2 2009-03-03 15hr 01min-set2.jpg|600px]] *Upper: Test by primer set1 (1-1 -> 8-1, ladder, 9-1 -> 16-1, ladder, 17-1 -> 22-1, X-1, Y-1) *Lower: Test by primer set2 (1-2 -> 8-2, ladder, 9-2 -> 16-2, ladder, 17-2 -> 22-2, X-2) *Primer name (amplicon size) 1-1 (270) 4-1 (383) 7-1 (242) 10-2 (299) 13-2 (1102) 16-2 (1324) 19-1 (1007) 22-2 (1166) 1-2 (676) 4-2 (896) 7-2 (498) 10-1 (214) 13-1 (679) 16-1 (806) 19-2 (1512) 22-1 (757) 2-1 (540) 5-1 (413) 8-2 (231) 11-1 (131) 14-1 (598) 17-2 (1240) 20-1 (174) x-2 (839) 2-2 (649) 5-2 (530) 8-1 (230) 11-2 (525) 14-2 (756) 17-1 (419) 20-2 (584) x-1 (147) 3-1 (235) 6-2 (1050) 9-1 (373) 12-1 (984) 15-1 (297) 18-2 (288) 21-1 (200) y-1 (122) 3-2 (1323) 6-1 (299) 9-2 (907) 12-2 (1689) 15-2 (1422) 18-1 (171) 21-2 (346) ==Discussion & Suggestion== *Except primer set1 chromosome X and Y which have only weak bands, all primers generate strong band with good amplification at 52C *The PCR cycle is too many -> saturation of amplicons *The primer concentration used in PCR is too high -> serious primer dimer seen on the gel
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