Editing
Hosuk:LabNotesMDA/2013-6-3
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!
====Actions for next run==== =====To get larger fragment size===== *This is the first priority *Reduce mixing speed, with lower ALS concentration and longer denature time *Jeffβs suggestion : 133mM of ALS after mixing with cell. *Mixer volume/ (Mixer volume β cell capture volume) = 1.12 --> Required concentration of KOH in prepaired ALS tube for 133mM = '''148.96 mM''' (~ 149mM) {| {{table}} | || | ALS, current || | ALS, new || |- |H2O || |69.5 uL || |74.52 uL || |- |1M DTT || |10 uL || |10 uL |- |5M KOH || |8 uL || |2.98 uL |- |8% Tween20 || |12.5 uL || |12.5 uL |} {| {{table}} | || | NS, current || | NS, new || |- |H2O || |0 uL || |54.9 uL || |- |NS stock || |87.5 uL || |32.6 uL |- |8% Tween20 || |12.5 uL || |12.5 uL |} *NS stock : 4mL of 1M HCl + 6mL of 1M TrisHCl =====To reduce sample loss===== *Push the volume in ring mixer with air, and use MDA master mix filled in mixer and run MDA *For the next device design, add inlet between mixer and compartment for extracting MDA product from ring mixer *Total Compartments volume will be almost matched with the volume of ring mixer, as usual. =====To minimize bias===== *Adjust (decrease) concentration of phi29 and primers. =====Changes for next device design===== *Add inlet channels between mixer and compartment, and one more inlet next to ALS for air input *Slightly increase mixer volume --> will check how much volume will be transferred from current ring mixer to compartments using actual ALS with 10min. mixing ====Test new ALS and longer Mixing, and volume transfer by air==== =====Cell lysis test with new ALS recipe===== *HeLa cell was used prior to using PGP1. *1 sec valving period --> 5 times slower than usual *Cell was not lysed immediately, but after 1 cycle in ring mixer, cell began to be lysed [[Media:newALS-149mM_HeLaCell_cap_small.wmv|Movie]] =====Volume transfer from Mixer to Compartment by pushing Air after 10min. mixing===== *About 3 compartment volumes were not filled. *ALS+Cell solution was remained on the wall of ring mixer. [[Media:TransferVolumefromMixertoCompWithAirAfter10minMixing.wmv|Movie]] *For the next real run, just filling compartment as much as possible, and run rest of procedure. **First check point is getting bigger fragment by reducing sheer stress. *And another option is **Close valves in between compartment, then fill water in ring mixer **Then fill the empty compartment, so that remaining of mixer could be flushed and go to empty compartments. ====MDA Run with PGP1F (3rd)==== *Same procedure with [[EricChu:LabNotesMDA/2013-5-20|Eric's LabNote @ 05/20]], '''EXCEPT''' **ALS mixing time --> 10min. with 1 sec valving period (previously 200ms) **Push the ALS+cell in mixer to compartment with air (start from 1.8psi, and 2.5psi at the end) **Because of remaining in mixer, first (left) 6 compartment were empty (I filled solution at the last compartment, so chamber 1~6 became empty) *Run MDA for 10hr.
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)
Template used on this page:
Template:Table
(
edit
)
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