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

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>Djacobse
>Djacobse
(Undo revision 52549 by Djacobse (talk))
Line 10: Line 10:


  1. Reaction Table
  1. Reaction 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="45" | &nbsp;
| width="65" | 10 nM OligoPool
| width="65" | 2X Kapa SYBR qPCR MM
| width="65" | 100 uM AP1V61U-Amino
| width="65" | 100 uM AP2V6
| width="65" | H20
| width="65" | Total Volume
|- style="background-color:#8DB4E2;font-size:12pt" align="center"
| height="15" | per rxn
| align="center" | 0.4
| align="center" | 50
| align="center" | 0.4
| align="center" | 0.4
| align="center" | 48.8
| align="center" | 100
|- style="font-size:12pt" align="center"
| height="30" | BSPP 1 (12.5x)
| align="center" | 5
| align="center" | 625
| 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" | 625
| align="center" | 5
| align="center" | 5
| align="center" | 610
| align="center" | 1250
|}
2. Aliquot 100 uL into each of 12 qPCR tubes
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 x17
    vii. 72C 2 min
    viii. 16C hold
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
===qPCR Results===
[[Image:2013-10-22-BSPPControl.png|600px]]
===Nanodrop Results===


{| 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"
|- style="font-size:12pt;font-weight:bold" align="center"
| width="65" height="15" | Sample
| width="65" height="15" | Probe
| width="65" | BSPP 1A
| width="65" | BSPP 1A
| width="65" | BSPP 1B
| width="65" | BSPP 1B
Line 35: Line 94:


|- style="font-size:12pt" align="center"
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ug in 35 uL
|style="font-weight:bold" height="15" | ug in 40 uL
| align="center" | 3.4
| align="center" | 3.9
| align="center" | 3.8
| align="center" | 3.9
| align="center" | 3.7
| align="center" | 3.6
| align="center" | 3.7
| align="center" | 3.6
| align="center" | 3.3
| align="center" | 3.3
| align="center" | 3.4
| align="center" | 3.2
| align="center" | 3.2
| align="center" | 3.2
| align="center" | 3.2
| align="center" | 2.9
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="30" | uL 10X buffer
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 5
|- 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
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | uL nfH2O
| align="center" | 8
| align="center" | 8
| align="center" | 8
| align="center" | 8
| align="center" | 8
| align="center" | 8
| align="center" | 8
| align="center" | 8


|- style="font-size:12pt" align="center"
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | Total
|style="font-weight:bold" height="15" | Total
| align="center" | 50
| align="center" | &nbsp;
| align="center" | 50
| align="center" | &nbsp;
| align="center" | 50
| align="center" | &nbsp;
| align="center" | 50
| align="center" | 15.2
| align="center" | 50
| align="center" | &nbsp;
| align="center" | 50
| align="center" | &nbsp;
| align="center" | 50
| align="center" | &nbsp;
| align="center" | 50
| align="center" | 14.2


|}
|}


2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; Continued [[Daniel:Notebook/HiResChrPaint/2013-10-23|tomorrow]]
==DpnII Digestion==

Revision as of 01:09, 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
  10 nM OligoPool 2X Kapa SYBR qPCR MM 100 uM AP1V61U-Amino 100 uM AP2V6 H20 Total Volume
per rxn 0.4 50 0.4 0.4 48.8 100
BSPP 1 (12.5x) 5 625 5 5 610 1250
BSPP 2 (12.5x) 5 625 5 5 610 1250
2. Aliquot 100 uL into each of 12 qPCR tubes 
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 x17
    vii. 72C 2 min
    viii. 16C hold
4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total)
5. Column Purification, elute with 40 uL EB
6. Measure concentrations in nanodrop

qPCR Results

File:2013-10-22-BSPPControl.png

Nanodrop Results

Probe 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 40 uL 3.9 3.8 3.9 3.7 3.6 3.7 3.6 3.3
Total       15.2       14.2

DpnII Digestion