*LEVEL-1
DNA Sequencing Request Form
Protein & Nucleic Acid Chemistry Laboratory
(Hodgkin Building, Lab 204 / Enquiries on ext.- 5576/5613/5531)
NAME:
DATE:
DEPARTMENT:
LAB No:
CHARGE CODE:
ORDER No:
Email Address:
TEL (Ext.):
* To be filled in for samples submitted as overnight cultures for Qiagen-Robot plasmid preparation. You supply cell pellets with
accompanying primers. See notes on reverse before submitting samples.
Label all tubes with : Sample/primer name, Lab No. & your initials.
No
Sample
Name
(note 1)
Plasmid
Name
Plasmid
size
(bp)
Insert
size
(bp)
High/
low
copy
no.
Host
Strain
( note 2)
O.D.
at
harvest
Primer
Name
(note 3)
Tm
°C
PNACL
Use
Only
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
OTHER INFORMATION (Please tick appropriate boxes)
•
Plasmid Type (pUC, pGEM, etc.) :
•
Is sequence G/C or A/T rich?:
•
Data to be supplied on :
•
Do you want excess DNA returned to you? YES...
•
Do you wish to use standard primer supplied by PNACL:
G/C....
CFS...
,
,
A/T...
or
E-mail…
NO...
(We only store for 1 month)
(See notes on other side)
PLEASE SEE OTHER SIDE FOR INSTRUCTIONS.... ——>
Level 1: AUG 07 version
NOTES
(1). Sample name is what will ultimately label your DNA sequence file. Maximum 8 characters.
(2). Growth condition • LB medium (Terrific broth and other rich media should be avoided)
• Cells should be grown to OD600nm of 1.5 to 4 units- typically for 12-16 hours
• If possible, use vectors with high copy numbers e.g. pUC / Bluescript / pGEM
• Host strain - avoid JM100 series, TG1 & TG2 - as these strains contain high carbohydrate level.
• Spin down cells in 16mm x 100mm round bottomed tubes. We can not accept pellets in other
dimension tubes.
(3). Please supply primer: Volume = 10µl @ Concentration = 0.8-1.0 pmol / µl.
Primers should be at least 18 bases long with a Tm of 55-60oC and have sequences without runs of
identical bases or self complimentarity.
PNACL has stocks of the following primers to be used with appropriate templates, free of charge.
If you wish to use these, please indicate by ticking the boxes next to the primers.
Primer
Sequence
M13 Forward
TGT AAA ACG ACG GCC AGT
M13 Reverse
CAG GAA ACA GCT ATG ACC
T7
AAT ACG ACT CAC TAT AGG G
T3
ATT AAC CCT CAC TAA AGG G
T7 terminator
TAT GCT AGT TAT TGC TCA GCG G
pGEX5'
GGG CTG GCA AGC CAC GTT TGG TG
pGEX3'
CCG GGA GCT GCA TGT GTC AGA GG
SP6
CAT ACG ATT TAG GTG ACA CTA TAG
BGHR
TAG AAG GCA CAG TCG AGG
KS
CCT CGA GGT CGA CGG TAT CG
SK
GCC GCT CTA GAA CTA GTG GAT C
CHAROMID F
GAA TTC GAG CTC GGT ACC C
CHAROMID R
AAG CTT GCA TGC CTG CAG
Please Tick
More information
see PNACL website: http://www.le.ac.uk/mrctox/pnacl
Level 1: AUG 07 version