Editing
Jeff:LabNotes/Microbiome/2009-9-23
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!
== '''Extraction of 500 bp plasmid insertion''' == *The biomiga miniprep II kit was used for extraction *The product was run on a 1 % agarose gel for 40 min *No restriction was used yet since it needs to be ordered '''Results''' The gel shows a similar band as the previous gel. Thus, a restriction enzyme must be used in order for the DNA to properly move through the gel. [[Image:9-23-09 gel.jpeg|400px]] == '''MDA in an Agarose base''' == ''Try a new technique for MDA by keeping agarose warm and adding last.'' '''Procedure''' *Filter LMT agarose using 5 um pores *Perform a serial dilution of bacteria in .75% LMT agarose to 1250 cell/uL. Also dilute in water for + control. *Scale up the volume for MDA so that it 3x a normal reaction so the agarose is easier to work with *Since 10 uL of template will be used, no water will be used in the master mix to keep the concentrations constant *Want to vary the volumes of beads to see which works best *The following volumes were used {| {{table}} | align="center" style="background:#f0f0f0;"|'''Reagent''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' |- | H20||0||0||0||0||0||0||0 |- | .75% LMT Agarose||12.6||12.1||11.6||11.1||10.6| | C12 bound to bead||0||0.5||1||1.5||2| | N6 free primer||3||3||3||3||3| | Buffer||6||6||6||6||6| | NTPs||2.4||2.4||2.4||2.4||2.4| | SYBR Green 2X||3||3||3||3||3| | Phi 29 Enzyme||3||3||3||3||3| | Total||30||30||30||30||30| | |} *Add all reagents to master mix except for agarose and enzyme *Keep agarose warmed and in its liquid phase. After all reagents are added besides the enzyme, add the agarose *Heat up bacteria at 90 C for 2 min to lyse. *While heating up, add Enzyme to master mix *Cool bacteria at 40 C for 2 min and then put on ice *While cooling at 40 C, mix thouroughly *Add 10 uL of bacteria in agarose to 8 tubes and 10 uL .75% agarose to 2 tubes (NTC). Put tubes on ice. *Add 10 uL of master mix to each respective tube. Final conc. of agarose is .375% *Perform real time MDA with the opticon machine '''Results''' The results show that the new method did not work. The agarose was probably too hot when added to the master mix and thus killed the enzyme. Furthermore, even though it was kept hot longer, it was still sticky and thus difficult to work with. I believe that we will have to find a better concentration of agarose to work with before we can continue because the MDA is too inconsistent. [[Image:9-23-09 mda.jpeg|400px]]
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