*LEVEL-2
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 for automated sequencing reaction set-up. You supply purified template DNA 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)
Template
name
(note 2)
Template
type &
size(bp)
(plasmid/PCR
product etc.)
Purification
method
Amount
Supplied
(ng DNA)
(note 2)
Quantification
method
(note 4)
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)
•
Is sequence G/C or A/T rich:
•
Data to be supplied on:
•
Do you wish to use standard primer supplied by PNACL:
G/C....
CFS...
,
A/T...
or
E-mail...
(See notes on other side)
PLEASE SEE OTHER SIDE FOR INSTRUCTIONS.....——>
Level 2: AUG 07 version
NOTES
(1). Sample name is what will ultimately label your DNA sequence data file. Maximum 8 characters.
(2). Amount of template. We require 8µl DNA per reaction containing appropriate amount of DNA. (If possible try to
send 9-10 l of the template per reaction) -Please follow these guidelines:
Template DNA
Plasmid
Size
3 - 10 kb
10 - 20 kb
Quantity
0.2-0.5 µg / 8µl
0.4-0.6 µg / 8µl
Cosmids
30 - 45 kb
0.2-0.4µg / 8µl
BACS
80 - 300 kb
0.25-0.5µg / 8µl
3 - 10 kb
100 - 200 bp
200 - 500 bp
500 - 1000 bp
1000 - 2000 bp
>2000bp
0.1µg / 8µl
1-3 ng / 8µl
3-10 ng / 8µl
5-20 ng / 8µl
10-40 ng / 8µl
20-50ng/8ul
M13 ssDNA
PCR products
(3). Please supply Primer: Volume = 10µl per reaction @ Concentration = 0.8-1.0 pmol / µl.
Primers should be at least 18 bases long with a T m 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
(4). Recommended quantification method is by agerose gel against known concentration of a Lamda ladder.
General 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.
More information
see PNACL website: http://www.le.ac.uk/mrctox/pnacl/
Level 2: AUG 07 version