Eccles:PCR Product Sequencing

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Sequencing Product Preparation

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

  1. 15 min @ 37°C
  2. 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

  1. 96°C 1:15 min
  2. 96°C 45 sec
  3. 50°C 45 sec
  4. 60°C 3:30 min
  5. return to step 2 - 24x

 

Clean up

2 ul 3M Sodium Acetate

10 ul dH2O

50 ul 95% ethanol

62 ul per reaction tube

 

  1. mix clean up solution with sample
  2. let sit 15 min
  3. 20 min spin 4 000 rpm
  4. decant and tap dry
  5. 200 ml 70% ethanol
  6. 10 min spin 4 000 rpm
  7. decant and tap dry
  8. repeat 5-7
  9. set upside down on a clean dry towel until dry, (~ an hour)
  10. add 15 ul high dye mix and re-suspend
  11. 95°C for 2 min and put on ice (burry the tubes)
  12. 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


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