Eccles:PCR Product Sequencing

       Sequencing Product Preparation



 Sequencing Product Preparation



 PCR clean up

 2 u l PCR product

 4  u <span style='font-size:10.0pt;font-family:Arial'> l dH2O

<p class=MsoNormal> 6 <span style='font-size:10.0pt;font-family:Symbol'>u <span style='font-size: 10.0pt;font-family:Arial'>l total (vary proportion based on PCR product conc’s)

<p class=MsoNormal>

<p class=MsoNormal> to each tube add; 1/2 u <span style='font-size:10.0pt;font-family:Arial'>l exo (exonuclease)

<p class=MsoNormal> 1/2 u <span style='font-size:10.0pt;font-family:Arial'>l sap (shrimp alkaline phosphatase)

<p class=MsoNormal> put on Pre-seq rxn

<ol style='margin-top:0cm' start=1 type=1> <li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt; font-family:Arial'>15 min @ 37 <span style='font-size:10.0pt; font-family:Symbol'>° <span style='font-size:10.0pt;font-family: Arial'>C </li> <li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt; font-family:Arial'>15 min @ 80 <span style='font-size:10.0pt; font-family:Symbol'>° <span style='font-size:10.0pt;font-family: Arial'>C </li> </ol>

<p class=MsoNormal> then into fridge to cool

<p class=MsoNormal>

Sequencing reaction add

<p class=MsoNormal> 0.64 <span style='font-size:10.0pt;font-family:Symbol'>u <span style='font-size: 10.0pt;font-family:Arial'>l primer

<p class=MsoNormal> 1 <span style='font-size:10.0pt;font-family:Symbol'>u <span style='font-size: 10.0pt;font-family:Arial'>l Big dye buffer

<p class=MsoNormal> 2 <span style='font-size:10.0pt;font-family:Symbol'>u <span style='font-size: 10.0pt;font-family:Arial'>l Big dye

<p class=MsoNormal>

Sequencing reaction

<ol style='margin-top:0cm' start=1 type=1> <li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt; font-family:Arial'>96 <span style='font-size:10.0pt;font-family: Symbol'>° C 1:15 min </li> <li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt; font-family:Arial'>96 <span style='font-size:10.0pt;font-family: Symbol'>° C 45 sec </li> <li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt; font-family:Arial'>50 <span style='font-size:10.0pt;font-family: Symbol'>° C 45 sec </li> <li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt; font-family:Arial'>60 <span style='font-size:10.0pt;font-family: Symbol'>° C 3:30 min </li> <li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt; font-family:Arial'>return to step 2 - 24x </li> </ol>

<p class=MsoNormal>

Clean up

<p class=MsoNormal> 2 <span style='font-size:10.0pt;font-family:Symbol'>u <span style='font-size: 10.0pt;font-family:Arial'>l 3M Sodium Acetate

<p class=MsoNormal> 10 <span style='font-size:10.0pt;font-family:Symbol'>u <span style='font-size: 10.0pt;font-family:Arial'>l dH2O

<p class=MsoNormal> 50 <span style='font-size:10.0pt;font-family:Symbol'> u <span style='font-size:10.0pt;font-family:Arial'> l 95% ethanol

<p class=MsoNormal> 62 <span style='font-size:10.0pt;font-family:Symbol'>u <span style='font-size: 10.0pt;font-family:Arial'>l per reaction tube

<p class=MsoNormal>

<ol style='margin-top:0cm' start=1 type=1> <li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt; font-family:Arial'>mix clean up solution with sample </li> <li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt; font-family:Arial'>let sit 15 min </li> <li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt; font-family:Arial'>20 min spin 4 000 rpm </li> <li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt; font-family:Arial'>decant and tap dry </li> <li class=MsoNormal style='color:black;tab-stops:list 36.0pt'><span style='font-size:10.0pt;font-family:Arial;color:windowtext'>200 <span style='font-size:10.0pt;font-family:Symbol;color:windowtext'>m <span style='font-size:10.0pt;font-family:Arial;color:windowtext'>l 70% ethanol </li> <li class=MsoNormal style='color:black;tab-stops:list 36.0pt'><span style='font-size:10.0pt;font-family:Arial;color:windowtext'>10 min spin 4 000 rpm </li> <li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt; font-family:Arial'>decant and tap dry </li> <li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt; font-family:Arial'>repeat 5-7 </li> <li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt; font-family:Arial'>set upside down on a clean dry towel until dry, (~ an    hour) </li> <li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt; font-family:Arial'> add 15 <span style='font-size:10.0pt; font-family:Symbol'>u <span style='font-size:10.0pt;font-family: Arial'>l high dye mix and re-suspend </li> <li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt; font-family:Arial'>95 <span style='font-size:10.0pt;font-family: Symbol'>° C for 2 min and put on ice (burry the tubes) </li> <li class=MsoNormal style='tab-stops:list 36.0pt'><span style='font-size:10.0pt; font-family:Arial'>send to Alison Fitches with information on the run time needed (see table below) </li> </ol>

<p class=MsoNormal>

<p class=MsoNormal> <br clear=ALL>