Editing
Alice:LabNotes/2008-9-3
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!
==Experiment: Extending the cosmic probe length== previous steps and the over-view of the experiment can be found through this link [[https://genome-tech.ucsd.edu/LabNotes/index.php/Alice:LabNotes/2008-8-25]] ==T7 exonuclease digestion== obtained 48 reactions tubes from Ida on cpg30K V2.2 1. The tubes were purified using ethanol purification, and diluted in 100ul H2O for every 24 reactions. 2. Add ratio of 0.5ul/tube amount of T7, which is 12ul of T7 and 11ul of NEBuffer #4 to each tube(24 reactions). 3. 25C for 2 hrs. 4. Qiaquick column purification, elute in 30ul of H2O. 5. Measure with Nanodrop. Nanodrop result: 125.8ng/ul TBE gel of the product after PCR and T7 digestion: [[Image:9-4-08.jpg]] ==circularization reaction== the concentration of the template from last step is 125.8ng/ul, so it is equivalent to 125.8/144/330=2.64uM reaction system 1: 1:1 ratio of template:circleHelper reaction system 2: 1:3 ratio of template:circleHelper reaction system 1: template (264nM) 2ul 10x Ampligase Buffer 2ul CircleHelper V4.2(1uM) 0.5ul dNTP(10mM) 0.5ul Stoffel fragment(10U/ul) 0.5ul Ampligase(5U/ul) 0.5ul ddH2O 14ul reaction system 2: template (264nM) 2ul 10x Ampligase Buffer 2ul CircleHelper V4.2(1uM) 1.5ul dNTP(10mM) 0.5ul Stoffel fragment(10U/ul) 0.5ul Ampligase(5U/ul) 0.5ul ddH2O 13ul 94C 2 min -> 55C 20 min TBU gel after the circularization reaction: [[Image:9-5-08.jpg]] add 1.5ul Exo I &III + 10ul of product from reaction system 1 37C 2hr -> 95 10min -> 4C Purify using Minelute columns 9/9/08: add 2.5ul Exo I&III + 20ul of product from reaction system 2 37C 2hr -> 95 10min -> 4C Purify using Minelute columns ==PCR (9/9/08)== H2O 40ul SYBR green (50x) 0.4ul Forward primer AmpRV6.2_2(100uM) 0.2ul Reverse primer AmpFV6.2_2(100uM) 0.2ul Taq master mix(2x) 50ul probe template (product after Exo I&III) 10ul 94 2min -> (94 30sec -> 55C 1min -> 72 45 sec)* 15 cycles -> 72C 5min -> 15C hold [[Image:9-9-08 PCR after Exo I&III.jpg]] TBU gel of each step:[[Image:9-9-08 after pcr.jpg]] ==T7 exonuclease digestion (9/9/08)== Add 2ul of T7 exonuclease to the PCR product 25C 2hr -> 4C Purify with Qiaquick columns, and measure the concentration using Nanodrop to determine the amount of spacer needed. Nanodrop result: 27.4ng/ul * 30ul ==Insert Spacer into Circularized DNA (9/10/08)== 27.4ng / (160*330) = 510nM, 5ul of template makes it 2.55 umol. 107bp spacer concentration is 156.7 ng/ul, which is 4.4 uM. We need to achieve 1:1 probe/spacer ratio, so the volume of spacer needed is 0.6 ul. template 5ul 10x Ampligase Buffer 2ul 107bp Spacer 0.6ul dNTP(10mM) 0.5ul Stoffel fragment(10U/ul) 0.5ul Ampligase(5U/ul) 0.5ul ddH2O 11ul (94C 2min -> 60C 20min ) x4 cycles add 2ul Exo I/III-> 37C 2h -> 94C 5mins >4C TBU gel of the results: [[Image:9-11-08 after spacer insertion.jpg]] ==PCR amplification after spacer insertion== H2O 70.6ul 10x Stoffel buffer 10ul MgCl2(25mM) 6ul dNTP(10mM each, NEB) 2ul Primer mix 100uM (Ap1V4IU +Ap2V4) 0.4ul 50x SYBG I 0.4ul template(from last step) 10ul Jumpstart Taq 0.6ul 94c 2min -> ( 94c 30sec -> 60C 1.5min -> 72C 1min-> Plate read -> ) x35 cycles -> 72C 5min -> 15C hold TBE gel after PCR: [[Image:9-11-08 PCR after spacer insertion.jpg]] [[Image:ZhangLab_2 2008-09-12 10hr 37min.jpg]]
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