Daniel:Notebook/HiResChrPaint/2013-5-10: Difference between revisions

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  learned from the previous tries is that I need more probe.  As such, I am modifying the qPCR.  First of all I'm going to use, on Dr. Zhang's  
  learned from the previous tries is that I need more probe.  As such, I am modifying the qPCR.  First of all I'm going to use, on Dr. Zhang's  
  recommendation, the suggested protocol for qPCR using the KAPA kit.  Previously I was using a different protocol that I felt was a bit short.
  recommendation, the suggested protocol for qPCR using the KAPA kit.  Previously I was using a different protocol that I felt was a bit short.
Also, in previous expansion PCRs I have done 8 reactions of 100 uL each for each probe.  I'm going to use 12 for each in an effort to increase
yields.


==Expansion qPCR==
==Expansion qPCR==
1. Set up master reactions according to table
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:12pt" align="center"
|style="background-color:#FFFF00" align="center" width="65" height="30" | &nbsp;
|style="background-color:#8DB4E2;font-weight:bold" width="65" | per rxn
|style="font-weight:bold" width="65" | V4S3 (12.5x)
|style="font-weight:bold" width="65" | V6S3 (12.5x)
|- style="font-size:12pt" align="center"
| height="30" | 20 nM OligoPool
|style="background-color:#8DB4E2" align="center" | 0.2
| align="center" | 2.5
| align="center" | 2.5
|- style="font-size:12pt" align="center"
| height="45" | 2X Kapa SYBR qPCR MM
|style="background-color:#8DB4E2" align="center" | 50
| align="center" | 625
| align="center" | 625
|- style="font-size:12pt" align="center"
| height="30" | 100 uM AP1V41U
|style="background-color:#8DB4E2" align="center" | 0.4
| align="center" | 5
| align="center" | 5
|- style="font-size:12pt" align="center"
| height="30" | 100 uM AP2V4
|style="background-color:#8DB4E2" align="center" | 0.4
| align="center" | 5
| align="center" | 5
|- style="font-size:12pt" align="center"
| height="30" | 100 uM AP1V61U
|style="background-color:#8DB4E2" align="center" | 0.4
| align="center" | 5
| align="center" | 5
|- style="font-size:12pt" align="center"
| height="30" | 100 uM AP2V6
|style="background-color:#8DB4E2" align="center" | 0.4
| align="center" | 5
| align="center" | 5
|- style="font-size:12pt" align="center"
| height="30" | 50 mM aa-dUTP
|style="background-color:#8DB4E2" align="center" | 0.1
| align="center" | 1.6
| align="center" | 1.6
|- style="font-size:12pt" align="center"
| height="15" | H20
|style="background-color:#8DB4E2" align="center" | 46
| align="center" | 575
| align="center" | 575
|- style="font-size:12pt" align="center"
| height="30" | Total Volume
|style="background-color:#8DB4E2" align="center" | 100
| align="center" | 850
| align="center" | 1250
|}
2. Aliquot 100 uL into each of 12 qPCR tubes (24 in total for both reactions)
3. PCR protocol:
    i. PCR 3 min 95C
    ii. 95C 3 sec
    iii. 55C 30 sec
    iv. 72C 20 sec
    v. plate read
    vi. goto ii
    vii. 72C 2 min
    viii. 16C hold
4. [[Daniel:Protocols/EtOHPrecip|Ethanol Precipitation]]

Revision as of 15:33, 10 May 2013

Probe Prep Test 2

Back to Calendar

Purpose: After extensive troubleshooting, I think I am once again ready to try creating the probes, starting from qPCR and hopefully
going all the way to staining.  My plan is to use only two sets, V4S3 and V6S3 as before, to help me focus better.  The key thing that I've
learned from the previous tries is that I need more probe.  As such, I am modifying the qPCR.  First of all I'm going to use, on Dr. Zhang's 
recommendation, the suggested protocol for qPCR using the KAPA kit.  Previously I was using a different protocol that I felt was a bit short.
Also, in previous expansion PCRs I have done 8 reactions of 100 uL each for each probe.  I'm going to use 12 for each in an effort to increase
yields.

Expansion qPCR

1. Set up master reactions according to table
  per rxn V4S3 (12.5x) V6S3 (12.5x)
20 nM OligoPool 0.2 2.5 2.5
2X Kapa SYBR qPCR MM 50 625 625
100 uM AP1V41U 0.4 5 5
100 uM AP2V4 0.4 5 5
100 uM AP1V61U 0.4 5 5
100 uM AP2V6 0.4 5 5
50 mM aa-dUTP 0.1 1.6 1.6
H20 46 575 575
Total Volume 100 850 1250
2. Aliquot 100 uL into each of 12 qPCR tubes (24 in total for both reactions)
3. PCR protocol:
    i. PCR 3 min 95C
    ii. 95C 3 sec
    iii. 55C 30 sec 
    iv. 72C 20 sec
    v. plate read
    vi. goto ii
    vii. 72C 2 min
    viii. 16C hold

4. Ethanol Precipitation