Editing
Noi/NOTES/2014-3-2
(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!
== PCR Amplification (TEST) == {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''12.5x rxn mix''' |- | Captured template||4.00||0.00 |- | 10uM AmpF6.4Sol||0.50||6.25 |- | 10uM AmpR6.3Indx1||0.50||6.25 |- | 2X KAPA SYBR MM||12.50||156.25 |- | H2O||7.50||93.75 |- | Total||25.00|| |} - Aliquot 21ul, add 4ul of circularized DNA<br> '''Program (Eppendorf Realplex)''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min ==== qPCR curves ==== [[File:2014-03-05_qPCR_TEST_MONOD_capture.png| 600px]] ==== Verified amplified products in 6% TBE gel ==== * Loaded 3ul of PCR product in 6% TBE gel [[File:ZhangLab_2 2014-03-05 13hr 23min_TEST_PCR_MONOD_MN_1-8.jpg|450px]] [[File:ZhangLab_2 2014-03-05 13hr 23min_TEST_PCR_MONOD_MN_9-11.jpg|260px]] * If everything works well, I will set up duplicate reactions of 50ul with 20% (v/v) of template {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''25x rxn mix''' |- | Captured template||10.00||0.00 |- | 10uM AmpF6.4Sol||1.00||25.00 |- | 10uM AmpR6.3IndX(X=81-92)||1.00||0.00 |- | 2X KAPA SYBR MM||25.00||625.00 |- | H2O||13.00||325.00 |- | Total||50.00|| |} - Aliquot 39ul, add 1ul of 10uM AmpR6.3IndX, add 10ul of circularized DNA<br> '''Program (Eppendorf Realplex)''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min ==== qPCR curves ==== I took the average of the replicates [[File:2014-03-05_qPCR_MONOD_capture.png| 600px]] : - Stopped MN_1-8 at 14 cycles : - Stopped MN_9-11 at 18 cycles : - Pool and purify PCR product with 0.7 volume of AMPure beads : - Eluted with 65ul H2O : - Quantify concentration in 6% TBE gel (load 1.5ul of purified libraries) :[[File:ZhangLab_2 2014-03-05 17hr 20min_MONOD_SeqLib_PQind.jpg|450px]] {| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Original IDs''' | align="center" style="background:#f0f0f0;"|'''Indexes''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount in 65ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 50ng (ul)''' |- | MN#1||BXPC3||Ind81||2.16||140.17||23.19 |- | MN#2||U87MG||Ind82||3.14||204.13||15.92 |- | MN#3||PANC1||Ind83||6.28||408.34||7.96 |- | MN#4||BE(2)C||Ind84||3.76||244.61||13.29 |- | MN#5||T98G||Ind85||1.90||123.48||26.32 |- | MN#6||H9 p43 BMP4||Ind86||8.26||536.74||6.06 |- | MN#7||H1 p47 Control||Ind87||2.77||180.10||18.05 |- | MN#8||H1 p47 BMP-4||Ind88||1.08||70.34||46.20 |- | MN#9||UCLA-B-F10||Ind89||3.32||216.00||15.05 |- | MN#10||UCLA-B-G10||Ind90||5.65||367.18||8.85 |- | MN#11||UCLA-B-G11||Ind91||1.50||97.31||33.40 |} : - Pool equal amount of sequencing libraries and performed PAGE size-selection in 2 of 5-well 6% TBE gels at the band ~375bp : [[File:ZhangLab_2 2014-03-05 18hr 59min_PAGE-SS.jpg| 500px]] : - Elute with 24ul H2O. : - Verify sequencing libraries in 6% TBE gel by loading 2ul : [[File:ZhangLab_2 2014-03-06 18hr 21min_verify_MONOD-SeqLib.jpg| 100px]] : -Hand to Alan for concentration quantification by qPCR '''Sequencing ibrary IDs: NP-BSPP-MONOD_81-91_Mar2, 2014'''<br> '''Sequencing run: ''' Dr. Zhang suggested to do MiSeq PE run, 150+7+150 bp : - MiSeq run: '''MONOD_81_91''' on March 3rd, 2014'''
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