Ordering of CEQ 2000 sequencing reageants:

For Template purification: PCR- Qiagen product- QIAquick PCR purification kit Cat. No. 28104 (50 tubes) 28106 (250 tubes) Plasmids- Promega wizard plasmid preps work well

For orders from Beckman use clark's account: DNA sequencing reagents: CEQ 2000 Dye terminator cycle sequencing with Quick stark kit, from Beckman Cat # 608120, for 100rxn, we do quarter rxns, so for 400 rxns. ~370$w/discount. ** Does not contain seq rxn buffer**

CEQ separation gel 1, cat # 608010 CEQ separation buffer, cat # 608012

For Template prep. Amount of DNA: dsDNA 25-100fmol ssDNA 25-50fmol Purified PCR product 25-100fmol *For fmol conversions see quickstart kit insert in sequencing folder.

For PCR purification: Two 50ul PCR rxns were combined and purified using the quiagen kit. DNA should be run on a gel and quantified. Eluted in 30ul ddH2O, used 2ul for sequencing rxn. *I had no success ethanol precipitating my template, while others reported that it worked*

For plasmid purification: 3mls of O.N. culture should be minipreped using the wizard preps. DNA should be eluted in 50ul (~0.2ug/ul).

CEQ 2000 Sequencing Protocol 1. Preparation of DNA Sequencing Reaction Located in -80 fridge, 2nd shelf from the top, 3rd tray from the left, first row of boxes. Labeled CEQ sequencing reagents. Prepare sequencing reaction in 0.2mL thin wall tube. (PCR drawer, Keep on ice) For quarter rxns add reagents, in the following order. 1. X µl of Template + X ddH20 = 10ul Usually use 2ul of template + 8ul ddH2O, but might need more depending upon size and concentration of template. 3. 2ul primer ~ 10uM (10pmol/ul) can be more/or less 100uM-1uM 4. 2ul seq mix (10X buffer, dNTP’s, ddNTP’s dye terminator, polymerase, aliquots are numbered use #1 first, ect.) 2. X amount of 1x seq rxn buffer (2-10ul) (buffer in orange lid is 10x, in sequencing box -80) 20 µl total

2. Thermal Cycling Program 96°C 20sec, 50°C 20 sec, 60°C 4 min, for 35 (can go higher, 40-50) cycles followed by holding at 4°C

3. Ethanol Precipitation a) to each tube add 4µl of freshly made Stop Solution [1.5M NaOAc, 50mM EDTA], (1:1 mix from 3M NaOAc and 100mM EDTA) and add 1µl of 20 mg/ml glycogen (in -80) Mix (found in -20 #1 in sequencing ER box) b) add 60µl 95% (v/v) EtOH, spin 15 at 4°C (-20 fridge #1) c) rinse 2X’s with 200µl of 70% EtOH spin 2 min 14,000rpm (-20 fridge #1) d) vacuum dry 30-40 min (NO HEAT) e) resuspend in 40µl of deionized formamide- labeled SLS for sequencing loading solution found in -80 in seq box, or can obtain from clark/shaeffer's lab). Add one to two drops of mineral oil. This will take 5-15 min. The resuspended samples can be stored in closed tubes at -20°C for up to one month. DO NOT STORE SAMPLES AS DRY PELLETS!

4. Sample Loading into the CEQ Gel tube and buffer are located in the middle drawer in fridge 3 Buffer loading trays are in the PCR drawer. open CEQ Seq open “Sample Setup Menu” go to >File >New – add Names choose method (Should say on machine which method is appropriate for corresponding capillaries) >File >Save – date eg. 090601 + name OK –all blue = Save go to “Run Menu” - close, DON’T Click OK for gel cartridge - direct Control Tab - click on Gel - insert Gel - Done (Event Type load Gel Cartridge) - Load Unload Plates (lower left Corner) leave on wetting tray -click “unload” - add Buffer Tray - samples - replace distil water in wetting tray - click load - go back to “sample setup” > run >start put in name for operator (let run) 5. Shutting Down go to “Run Menu” Direct Control Gel ask “do you want to release” OK, Not Installed - lower left – load/unload plates “unload” - remove loading buffer plates and samples - clean wetting tray and replace with new distilled water - Load

6. Retrieving Sequence From 614 On their computers First export the files from Seq manager File>Export> Save to 'lai' folder which is shared

On our computers: Network Neighbor>Entire Network>Microsoft windows network>614 Mueller>Then depending upon which seq computer you used chose the computer. To log into the computer: For DNASeq Connect as: DNASeq Password: DNASeq

For DNASeq2 Connect as: DNASeq2 Password: DNASeq2 (Case Sensitive)