*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