Eccles:PCR Product Sequencing
<html>
<head> <meta name=Title content="Sequencing Product Preparation"> <meta name=Keywords content=""> <meta http-equiv=Content-Type content="text/html; charset=macintosh"> <meta name=ProgId content=Word.Document> <meta name=Generator content="Microsoft Word 10"> <meta name=Originator content="Microsoft Word 10"> <link rel=File-List href="PCR%20Product%20Sequencing_files/filelist.xml"> <title>Sequencing Product Preparation</title> <style> </style> </head>
<body bgcolor=white lang=EN-US style='tab-interval:36.0pt'>
Sequencing Product Preparation
PCR clean up
2 ul PCR product
4 ul dH2O
6 ul total (vary proportion based on PCR product conc’s)
to each tube add; 1/2 ul exo (exonuclease)
1/2 ul sap (shrimp alkaline phosphatase)
put on Pre-seq rxn
- 15 min @ 37°C
- 15 min @ 80°C
then into fridge to cool
Sequencing reaction add
0.64 ul primer
1 ul Big dye buffer
2 ul Big dye
Sequencing reaction
- 96°C 1:15 min
- 96°C 45 sec
- 50°C 45 sec
- 60°C 3:30 min
- return to step 2 - 24x
Clean up
2 ul 3M Sodium Acetate
10 ul dH2O
50 ul 95% ethanol
62 ul per reaction tube
- mix clean up solution with sample
- let sit 15 min
- 20 min spin 4 000 rpm
- decant and tap dry
- 200 ml 70% ethanol
- 10 min spin 4 000 rpm
- decant and tap dry
- repeat 5-7
- set upside down on a clean dry towel until dry, (~ an hour)
- add 15 ul high dye mix and re-suspend
- 95°C for 2 min and put on ice (burry the tubes)
- send to Alison Fitches with information on the run time needed (see table below)
|
Bases |
Time (min) |
|
< 200 |
75 |
|
200-250 |
80 |
|
251-290 |
85 |
|
291-320 |
90 |
|
321-350 |
95 |
|
351-380 |
100 |
|
381-410 |
110 |
|
411+ |
120 |
</body>
</html>
