*LEVEL-1DNA 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?: G/C.... , A/T...

•Data to be supplied on : CFS..., orE-mail…

•Do you want excess DNA returned to you? YES... NO... (We only store for 1 month)

•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 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.

PrimerSequencePlease Tick

M13 Forward TGT AAA ACG ACG GCC AGT

M13 Reverse CAG GAA ACA GCT ATG ACC

T7AAT ACG ACT CAC TAT AGG G

T3ATT AAC CCT CAC TAA AGG G

T7 terminatorTAT 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

SP6CAT ACG ATT TAG GTG ACA CTA TAG

BGHRTAG AAG GCA CAG TCG AGG

KSCCT CGA GGT CGA CGG TAT CG

SKGCC GCT CTA GAA CTA GTG GAT C

CHAROMID FGAA TTC GAG CTC GGT ACC C

CHAROMID RAAG CTT GCA TGC CTG CAG

More information

see PNACL website:

Level 1: AUG 07 version