Daniel:Notebook/HiResChrPaint/2013-10-3: Difference between revisions

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The control sample worked well enough, it seems like there is plenty of signal but it isn't in any particular place.  The experiment group, however, looked terrible, as it looked like there were no cells anywhere on the dish.  Further, there were scratches everywhere, distorting the view.  I'll have to make sure on future samples that the cells, after fixation, look healthy and plentiful.
The control sample worked well enough, it seems like there is plenty of signal but it isn't in any particular place.  The experiment group, however, looked terrible, as it looked like there were no cells anywhere on the dish.  Further, there were scratches everywhere, distorting the view.  I'll have to make sure on future samples that the cells, after fixation, look healthy and plentiful.
=V6S3/V4S1 Probe Production (Amino primers)=
Since I'm out of V6S3 and V4S1 I'm going to make some more.
==qPCR Amplification==
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" | 20 nM OligoPool
| width="65" | 2X Kapa SYBR qPCR MM
| width="65" | 100 uM AP1V41U-Amino
| width="65" | 100 uM AP2V4
| 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="30" | std 100 uL rxn
| align="center" | 0.2
| align="center" | 50
| align="center" | 0.4
| align="center" | 0.4
| align="center" | 0.4
| align="center" | 0.4
| align="center" | 46.5
| align="center" | 100
|- style="font-size:12pt" align="center"
| height="30" | V4S1 (12.5x)
| align="center" | 2.5
| align="center" | 125
| align="center" | 5
| align="center" | 5
| align="center" | 0
| align="center" | 0
| align="center" | 581.25
| align="center" | 1250
|- style="font-size:12pt" align="center"
| height="30" | V6S3 (12.5x)
| align="center" | 2.5
| align="center" | 125
| align="center" | 0
| align="center" | 0
| align="center" | 5
| align="center" | 5
| align="center" | 581.25
| align="center" | 1250
|}
2. Aliquot 100 uL into each of 12 qPCR tubes (24 in total)
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. Pool 3 samples together (300 uL each)
5. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB
6. Measure in nanodrop
===Nanodrop Results===
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" width="65" height="15" | &nbsp;
|style="font-weight:bold" width="65" | V4S1A
|style="font-weight:bold" width="65" | V4S1B
|style="font-weight:bold" width="65" | V4S1C
|style="font-weight:bold" width="65" | V4S1D
|style="font-weight:bold" width="65" | V6S3A
|style="font-weight:bold" width="65" | V6S3B
|style="font-weight:bold" width="65" | V6S3C
|style="font-weight:bold" width="65" | V6S3D
|- style="font-size:12pt" align="center" valign="bottom"
|style="font-weight:bold" height="15" | ng/uL
| align="center" | 16.2
| align="center" | 19.9
| align="center" | 16.1
| align="center" | 18.9
| align="center" | 92.2
| align="center" | 92.9
| align="center" | 98.2
| align="center" | 84.5
|- style="font-size:12pt" align="center" valign="bottom"
|style="font-weight:bold" height="15" | ug in 40 uL
| align="center" | 0.6
| align="center" | 0.8
| align="center" | 0.6
| align="center" | 0.8
| align="center" | 3.7
| align="center" | 3.7
| align="center" | 3.9
| align="center" | 3.4
|- style="font-size:12pt" align="center" valign="bottom"
|style="font-weight:bold" height="15" | total ug
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | 2.8
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | 14.7
|}
==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" | V4S1A
| width="65" | V4S1B
| width="65" | V4S1C
| width="65" | V4S1D
| width="65" | V6S3A
| width="65" | V6S3B
| width="65" | V6S3C
| width="65" | V6S3D
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ng/uL
| align="center" | 16.2
| align="center" | 19.9
| align="center" | 16.1
| align="center" | 18.9
| align="center" | 92.2
| align="center" | 92.9
| align="center" | 98.2
| align="center" | 84.5
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ug in 35 uL
| align="center" | 0.6
| align="center" | 0.7
| align="center" | 0.6
| align="center" | 0.7
| align="center" | 3.2
| align="center" | 3.3
| align="center" | 3.4
| align="center" | 3.0
|- 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/2013-10-4|tomorrow]]

Latest revision as of 00:04, 4 October 2013

FISH (Started 10/01/2013)[edit]

Back to Calendar

Microscopy[edit]

Used the Olympus Microscope upstairs to view the most recent round of FISH slides.

Results[edit]

Control (BSPP-2)

File:Overlay con 10x FISH2013 10 03.png File:Overlay con 40x FISH2013 10 03.png
10x magnification; BF,DAPI,TXRED 40x magnification; BF,DAPI,TXRED

Experiment (V6S1+V6S3)

File:Overlay exp 20x FISH 2013 10 03.png
20x magnification; BF,DAPI,TXRED

Discussion[edit]

The control sample worked well enough, it seems like there is plenty of signal but it isn't in any particular place. The experiment group, however, looked terrible, as it looked like there were no cells anywhere on the dish. Further, there were scratches everywhere, distorting the view. I'll have to make sure on future samples that the cells, after fixation, look healthy and plentiful.

V6S3/V4S1 Probe Production (Amino primers)[edit]

Since I'm out of V6S3 and V4S1 I'm going to make some more.

qPCR Amplification[edit]

1. Reaction Table
  20 nM OligoPool 2X Kapa SYBR qPCR MM 100 uM AP1V41U-Amino 100 uM AP2V4 100 uM AP1V61U-Amino 100 uM AP2V6 H20 Total Volume
std 100 uL rxn 0.2 50 0.4 0.4 0.4 0.4 46.5 100
V4S1 (12.5x) 2.5 125 5 5 0 0 581.25 1250
V6S3 (12.5x) 2.5 125 0 0 5 5 581.25 1250
2. Aliquot 100 uL into each of 12 qPCR tubes (24 in total)
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. Pool 3 samples together (300 uL each)
5. Column Purification, elute with 40 uL EB
6. Measure in nanodrop

Nanodrop Results[edit]

  V4S1A V4S1B V4S1C V4S1D V6S3A V6S3B V6S3C V6S3D
ng/uL 16.2 19.9 16.1 18.9 92.2 92.9 98.2 84.5
ug in 40 uL 0.6 0.8 0.6 0.8 3.7 3.7 3.9 3.4
total ug       2.8       14.7

DpnII Digestion[edit]

1. Reaction table
Sample V4S1A V4S1B V4S1C V4S1D V6S3A V6S3B V6S3C V6S3D
ng/uL 16.2 19.9 16.1 18.9 92.2 92.9 98.2 84.5
ug in 35 uL 0.6 0.7 0.6 0.7 3.2 3.3 3.4 3.0
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