Rosenblum_Lab_Sequencing

moredwarfBiotechnology

Oct 1, 2013 (4 years and 11 days ago)

89 views

1. PCR

2. PCR cleanup


Qiaquick PCR cleanup columns

3. Nanodrop cleaned PCR product, want to use 40
-
90ng PCR
-
DNA/rxn

4. Set up seq rxn


4 uL Big Dye mix (from WSU sequencing facility)


1 uL primer 10uM


q.s. DNA 50ng


q.s. 10uL total volume

5. seq rxn


5a
. 96 C 10 seconds


5b. 50 C 15 seconds


5c. 60 C 4 minutes (I lower this based on length of my PCR
product)


5d. go to 2, 24 times


5e. 4 C forever

6. Clean seq rxn using EdgeBio columns (from WSU sequencing
facility)


6a. Spin columns 3m 850g hinges out


6b. Transfer column to collection tube (labeled with seq
sheet name)


6c. Pipette entire seq rxn onto center of column


6d. Spin columns 3m 850g hinges out

7. Dry down rxn in vacufuge, ~ 20m V
-
Aq

8. Drop
-
off/mail samples with sample sheet (below, and avail
able
on WSU bioinformatics server under PI
-
folder, Downloads,
“DNASeqSampleSheet”.


Washington State University



Mailing Address:

Center for Integrated Biotechnology


100 Dairy Rd.

Bioinformatics Core Facility



Biotech. Life Sciences
Building

1715 NE S.
Fairway Rd.




Room 202

Room 227






c/o Derek Pouchnik

Pullman WA


99164




Pullman WA 99164
-
7520

(Manager,
Derek Pouchnik 509
-
335
-
1174)

Lab hours 8am
-
5pm


9. To retrieve sequences from core
-
data server


smb://bioinfo
-
serv1.ad.wsu.edu



usr: UI_user



ps
swrd: thAwrE5




PI_Folders




Off_Campus




UI_Rosenblum

Laboratory for Biotechnology and Bioanalysis Unit 1 (LBB1)


DNA Sequencing Sample Sheet


Date_____________







User Name________________


PI_______________







Phone_____________
_______











E
-
Mail___________________


Sample Code



Template




Primer



Special Notes

1.________

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23._
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25._______

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Data Printouts:

Yes
No




Clean
-
up:

LBB1 Spin Column

Yes No



Special

notes
: Lab Hours 8:00AM
-
5:00PM Monday
-
Friday

All data will be sent directly to the Bioinformatics server and will not be e
-
mailed. If you need help accessing the server please contact
the WSU Bioin
formatics Core Lab (
www.bioinformatics.wsu.edu
) or Justin Fischer(335
-
3744).

This form is also available on the Bioinformatics server PI folder under “DNASeqSampleSheet”.

Please keep sample codes as short
as possible…Thank You!!




BioTech Life Sciences Bldg, S.
Fairway