Daniel:Notebook/HiResChrPaint/2013-10-22: Difference between revisions

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{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:12pt;font-weight:bold" align="center"
| width="65" height="15" | Sample
| width="65" | BSPP 1A
| width="65" | BSPP 1B
| width="65" | BSPP 1C
| width="65" | BSPP 1D
| width="65" | BSPP 2A
| width="65" | BSPP 2B
| width="65" | BSPP 2C
| width="65" | BSPP 2D
|- style="font-size:12pt" align="center"
|- style="font-size:12pt" align="center"
|style="background-color:#FFFF00" align="center" width="65" height="45" | &nbsp;
|style="font-weight:bold" height="15" | ng/uL
| width="65" | 10 nM OligoPool
| align="center" | 97.9
| width="65" | 2X Kapa SYBR qPCR MM
| align="center" | 94.5
| width="65" | 100 uM AP1V61U-Amino
| align="center" | 96.9
| width="65" | 100 uM AP2V6
| align="center" | 91.9
| width="65" | H20
| align="center" | 90.2
| width="65" | Total Volume
| align="center" | 92.5
| align="center" | 90.7
| align="center" | 82.4


|- style="background-color:#8DB4E2;font-size:12pt" align="center"
|- style="font-size:12pt" align="center"
| height="15" | per rxn
|style="font-weight:bold" height="15" | ug in 35 uL
| align="center" | 0.4
| align="center" | 3.4
| align="center" | 50
| align="center" | 3.3
| align="center" | 0.4
| align="center" | 3.4
| align="center" | 0.4
| align="center" | 3.2
| align="center" | 48.8
| align="center" | 3.2
| align="center" | 100
| align="center" | 3.2
| align="center" | 3.2
| align="center" | 2.9


|- style="font-size:12pt" align="center"
|- style="font-size:12pt" align="center"
| height="30" | BSPP 1 (12.5x)
|style="font-weight:bold" height="30" | uL 10X buffer
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 625
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 610
| align="center" | 1250
|- style="font-size:12pt" align="center"
| height="30" | BSPP 2 (12.5x)
| align="center" | 5
| align="center" | 5
| align="center" | 625
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 610
| align="center" | 1250


|}
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="30" | uL DpnII (50U/uL)
| align="center" | 2
| align="center" | 2
| align="center" | 2
| align="center" | 2
| align="center" | 2
| align="center" | 2
| align="center" | 2
| align="center" | 2


2. Aliquot 100 uL into each of 12 qPCR tubes
|- style="font-size:12pt" align="center"
3. PCR protocol:
|style="font-weight:bold" height="15" | uL nfH2O
    i. PCR 3 min 95C
| align="center" | 8
    ii. 95C 3 sec
| align="center" | 8
    iii. 55C 30 sec
| align="center" | 8
    iv. 72C 20 sec
| align="center" | 8
    v. plate read
| align="center" | 8
    vi. goto ii x17
| align="center" | 8
    vii. 72C 2 min
| align="center" | 8
    viii. 16C hold
| align="center" | 8
4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total)
5. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB
6. Measure concentrations in nanodrop


===aqPCR Results===
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | Total
| align="center" | 50
| align="center" | 50
| align="center" | 50
| align="center" | 50
| align="center" | 50
| align="center" | 50
| align="center" | 50
| align="center" | 50
 
|}


[[Image:2013-10-22-BSPPControl.png|600px]]
2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; Continued [[Daniel:Notebook/HiResChrPaint/2013-10-23|tomorrow]]

Revision as of 01:05, 23 October 2013

BSPP Control

Back to Calendar

  • For first production of BSPP control, see 09/03/2013

Since I'm out of control group, I'm going to make some more.

qPCR

1. Reaction Table
Sample BSPP 1A BSPP 1B BSPP 1C BSPP 1D BSPP 2A BSPP 2B BSPP 2C BSPP 2D
ng/uL 97.9 94.5 96.9 91.9 90.2 92.5 90.7 82.4
ug in 35 uL 3.4 3.3 3.4 3.2 3.2 3.2 3.2 2.9
uL 10X buffer 5 5 5 5 5 5 5 5
uL DpnII (50U/uL) 2 2 2 2 2 2 2 2
uL nfH2O 8 8 8 8 8 8 8 8
Total 50 50 50 50 50 50 50 50
2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; Continued tomorrow