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

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>Djacobse
(Created page with "=BSPP Control= Back to Calendar *For first production of BSPP control, see 09/03/2013 Since I'm...")
 
>Djacobse
 
(4 intermediate revisions by the same user not shown)
Line 16: Line 16:
| width="65" | 10 nM OligoPool
| width="65" | 10 nM OligoPool
| width="65" | 2X Kapa SYBR qPCR MM
| width="65" | 2X Kapa SYBR qPCR MM
| width="65" | 100 uM AP1V61U
| width="65" | 100 uM AP1V61U-Amino
| width="65" | 100 uM AP2V6
| width="65" | 100 uM AP2V6
| width="65" | H20
| width="65" | H20
Line 63: Line 63:
  5. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB
  5. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB
  6. Measure concentrations in nanodrop
  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>
|- style="font-size:12pt;font-weight:bold" align="center"
| width="65" height="15" | Probe
| 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-weight:bold" height="15" | ng/uL
| align="center" | 97.9
| align="center" | 94.5
| align="center" | 96.9
| align="center" | 91.9
| align="center" | 90.2
| align="center" | 92.5
| align="center" | 90.7
| align="center" | 82.4
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ug in 40 uL
| 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
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | Total
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | 15.2
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | 14.2
|}
==DpnII Digestion==
1. Reaction Table
{| 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-weight:bold" height="15" | ng/uL
| align="center" | 97.9
| align="center" | 94.5
| align="center" | 96.9
| align="center" | 91.9
| align="center" | 90.2
| align="center" | 92.5
| align="center" | 90.7
| align="center" | 82.4
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ug in 35 uL
| align="center" | 3.4
| 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-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
|}
2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; Continued [[Daniel:Notebook/HiResChrPaint/2013-10-23|tomorrow]]

Latest revision as of 01:10, 23 October 2013

BSPP Control[edit]

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[edit]

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[edit]

File:2013-10-22-BSPPControl.png

Nanodrop Results[edit]

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[edit]

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