http://www.openwetware.org/index.php?title=Rutgers:Big_Dye_Sequencing&feed=atom&action=historyRutgers:Big Dye Sequencing - Revision history2013-05-25T07:42:21ZRevision history for this page on the wikiMediaWiki 1.13.2http://www.openwetware.org/index.php?title=Rutgers:Big_Dye_Sequencing&diff=574078&oldid=prevChristopher C Vanlang: New page: === Big dye sequencing reaction === 4.1- Equipment *PCR machine 4.2- Reagents 4.3- Other consumables 4.4- Procedure *All the following steps must be made on ice *Prepare the following m...2011-12-30T03:49:10Z<p>New page: === Big dye sequencing reaction === 4.1- Equipment *PCR machine 4.2- Reagents 4.3- Other consumables 4.4- Procedure *All the following steps must be made on ice *Prepare the following m...</p>
<p><b>New page</b></p><div>=== Big dye sequencing reaction ===<br />
4.1- Equipment<br />
*PCR machine<br />
<br />
4.2- Reagents<br />
<br />
4.3- Other consumables<br />
<br />
4.4- Procedure<br />
*All the following steps must be made on ice<br />
*Prepare the following mix (quantities are for 1 tube):<br />
- enhancer: 1 µl (if sequence GC rich)<br />
- big dye: 1 µl<br />
- primer M13: 0.33 µl (10 µM stock=3.3 pmol)<br />
- 5X buffer: 1.0 µl<br />
- water: 4.67 µl<br />
- cleaned template: 2 µl<br />
*put 8 µl of the mix in strip tubes<br />
*add 2 µl of template <br />
<br />
*Use a PCR machine for cycle sequecing (SWATI-SEQQ):<br />
lid temp: 110.0°C<br />
96°C: 10 sec<br />
50°C: 5 sec<br />
60°C: 4 min<br />
25°C: pause<br />
<br />
=== Cleaning of the sequencing reaction products by precipitation ===<br />
Best protocols would be under [[Purification of DNA]].<br />
<br />
General Procedure goes as follows<br />
#Add 1/10 volume of 3M sodium acetate and 2-3 volumes of 100% Ethanol<br />
#Mix and freeze overnight in -20. <br />
#Spin at full speed in a standard microcentrifuge at 4 degrees for 30 minutes.<br />
#Dry the pellet. <br />
#Add your desired quantity of water. Vortex and spin down to resuspend.<br />
<br />
=== Sequencing ===<br />
See [[Sequencing DNA]]<br />
<br />
6.1- Equipment<br />
*Applied Biosystems "3100-Avant Genetic Analyze"<br />
<br />
6.2- Reagents<br />
<br />
6.3- Other consumables<br />
*sequencing plate<br />
*septum<br />
<br />
6.4- Procedure<br />
*launch "3100 Avant data coll"<br />
*ignore request for ZIP disk<br />
*insert sample names (BLOCKS of four)<br />
*Dye set: "z"<br />
*Mobility file: DT3100POP6(BDv3)v1.mob<br />
*Project name: 3100-Avant project1<br />
*Run module: XXXXLongRun<br />
*Analysis module: BC-3100APOP6SR_SeqOffFtOff.saz<br />
*Click OK<br />
*place the plate on a base and a cover on top, press down<br />
*press "Tray"<br />
*put plate, press evenly down, close door<br />
*select "JPG", click plate image (goes from yellow to green)<br />
*click on the green triangle pointing to the right<br />
*go to status or run view</div>Christopher C Vanlang