AlanFung:LabNotes/Probe/2009-4-10: Difference between revisions

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(New page: ='''Production PCR for probe CPG_D40809 (24 wells)'''= =Objective= *Get familiar with the probe synthesis procedure *Optimization of Padlock Probe Preparation *Repeat PCR reaction setup a...)
 
>Alan6017518
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='''Production PCR for probe CPG_D40809 (24 wells)'''=


=Objective=
*Get familiar with the probe synthesis procedure
*Optimization of Padlock Probe Preparation
*Repeat PCR reaction setup and Probe purification with ethanol precipitation with optimized condition
==Samples & Materials==
*Jan_09_CPG_D40809 100K Oligo (10uM)
*Econo Taq
*AP1V6 AP2V6 Primer Mix (50uM)
*RNAse free H2O
*50X SYBG I
*3M NaOAC
*GlycoBlue
*100% Ethanol
=Overview=
*PCR reaction system setup
*Probe purification with ethanol precipitation
*Purify the reaction with Qiaquick columns
*Adapter Removal
*Page Denaturing Gel
*Purify the product from the gel
*Precipitation
*Quantification of DNA using denaturing gel and a quantitative ladder
=Procedure=
==Step 1: PCR reaction system setup==
*Thaw Econo Taq on ice
*Prepare strips and tubes on cooling rack
*Add in Content in table to tubes accordinly
{| border="1" align="center"
| align="center" style="background:#f0f0f0;"|'''Content'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''X24'''
| align="center" style="background:#f0f0f0;"|'''Tube A'''
| align="center" style="background:#f0f0f0;"|'''Tube B'''
|-
|Jan_09_CPG_D40809 (10uM)||align="right"|0.2uL||align="right"|4.8uL||align="right"|2.4uL||align="right"|2.4uL
|-
|Econo Taq||align="right"|50uL||align="right"|1250uL||align="right"|625uL||align="right"|625uL
|-
|AP1V6 AP2V6 Mix (50uM)||align="right"|0.4uL||align="right"|9.6uL||align="right"|4.8uL||align="right"|4.8uL
|-
|RNAse free H2O||align="right"|50uL||align="right"|1200uL||align="right"|600uL||align="right"|600uL
|-
|50X SYBG I||align="right"|0.4uL||align="right"|9.6uL||align="right"|4.8uL||align="right"|4.8uL
|-
|Total Volume|| align="right"| 101uL||align="right"|2424uL||align="right"|1212uL||align="right"|1212uL
|}
*Mix content in 2 x 1.5mL tube vortex and spin down
*Aliquot 100uL using Eppendorf repeat pipette to 24 wells (3 strips) on cooling rack
*Repeat pipette tip cannot fit into 1.5mL tube, change to P200 after 16 wells
*Seal strips with strip caps
*Place into Real Time PCR
*Perform
94C 2M > (94C 30S > 58C 30S > 72C 30S) X22 > 72C 3M > 15C Hold
*Stop reaction after cycles (Reached plateau)
==Step 2: Probe purification with ethanol precipitation==
*Add in required volume of 100% ethanol, 3M NaOAC and Glycoblue into tray
{| border="1" align="center"
| align="center" style="background:#f0f0f0;"|'''Content'''
| align="center" style="background:#f0f0f0;"|'''Volume Needed'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
|100% Ethanol||align="right"|2.5X Sample Volume||align="right"|6000uL
|-
|3M NaOAC||align="right"|0.1X Sample Volume||align="right"|240uL
|-
|Glycoblue||align="right"|1/300X Sample Volume||align="right"|8uL
|-
|Sample||align="right"|Sample Volume||align="right"|2400uL
|-
|Total Volume
|colspan="2" align="right"|8648uL
|-
|}
*Mix all content by repeat pipetting using multichannel pipette
*Pool all 24 wells PCR product into tray using multichannel pipette(P300)
*Mix all content by repeat pipetting using multichannel pipette
*Aliquot about 3750uL to 2 x 15mL tube
*Store in -80C
=Results=
=Suggestion=

Latest revision as of 22:54, 11 April 2009