*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) / Quantif-ication 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: G/C.... , A/T...

• Data to be supplied on: CFS... or E-mail...

• Do you wish to use standard primer supplied by PNACL: (See notes on other side)

PLEASE SEE OTHER SIDE FOR INSTRUCTIONS.....——>
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 ml of the template per reaction) -Please follow these guidelines:

Template DNA / Size / Quantity
Plasmid / 3 - 10 kb
10 - 20 kb / 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
M13 ssDNA / 3 - 10 kb / 0.1µg / 8µl
PCR products / 100 - 200 bp
200 - 500 bp
500 - 1000 bp
1000 - 2000 bp
>2000bp / 1-3 ng / 8µl
3-10 ng / 8µl
5-20 ng / 8µl
10-40 ng / 8µl
20-50ng/8ul

(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 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 Please Tick

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

(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