Daniel:Notebook/HiResChrPaint/2013-4-27: Difference between revisions

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[[Daniel:Notebook/HiResChrPaint|Back to calendar]]
[[Daniel:Notebook/HiResChrPaint|Back to calendar]]


==Ethanol Precipitation (Continued from [[Daniel:Notebook/HiResChrPaint/2013-4-26|4/26]]==
==Ethanol Precipitation (Continued from [[Daniel:Notebook/HiResChrPaint/2013-4-26|4/26/2013]])==


  5. Took samples from -80 and centrifuged at 14000 rpm for 25 minutes at 4 C
  5. Took samples from -80 and centrifuged at 14000 rpm for 25 minutes at 4 C
Line 91: Line 91:
  7. Loaded 12 uL each sample and ran gel for 20 minutes at 250V
  7. Loaded 12 uL each sample and ran gel for 20 minutes at 250V
  8. Stained with 3 uL SYBR gold for 25 minutes
  8. Stained with 3 uL SYBR gold for 25 minutes
===Gel Results===
Volume Report:  [[File:GelQuant_042713.xls]]
[[Image:GelQuant_042713.jpg|600px]]
[[Image:GelQuant_analysis_042713.jpg|600px]]
===Sample Concentrations===
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|-  align="center" valign="bottom"
| width="69" height="12" | Sample
| width="69" | ng/uL
| width="69" | ng (7 uL)
|-  align="center" valign="bottom"
| height="12" | V4S3
| align="center" | 4.6
| align="center" | 32.4
|-  align="center" valign="bottom"
| height="12" | V4S3
| align="center" | 3.1
| align="center" | 22.0
|-  align="center" valign="bottom"
| height="12" | V6S3
| align="center" | 3.5
| align="center" | 24.7
|}
==Discussion==
With the abysmally low yields from the Lambda Exonuclease reaction, I have come to the conclusions that it is this reaction causing the
  most problems in my probe prep protocol.  I'm going to need to approach this reaction better before I continue.  The following table
  summarizes the yields in (ug and %) comparing my probe preps and Noi's:
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:12pt" align="center"
| align="center" width="65" height="42" | &nbsp;
| width="65" | qPCR (ug)
| width="50" | qPCR Yield (%)
| width="65" | DpnII (ug)
| width="71" | DpnII Yield (%)
| width="65" | Lambda Exo (ug)
| width="65" | Lambda Exo Yield (%)
| width="65" | Yield from DPNII
|- style="font-size:12pt" align="center" valign="bottom"
|style="font-weight:bold" height="15" | Me
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | V4S3
| align="center" | 8.2
| align="center" | 100
| align="center" | 5.6
| align="center" | 68.3
| align="center" | 0.03
| align="center" | 0.4
| align="center" | 0.6
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | V6S3
| align="center" | 7.4
| align="center" | 100
| align="center" | 4.7
| align="center" | 63.5
| align="center" | 0.02
| align="center" | 0.3
| align="center" | 0.5
|- style="font-size:12pt" align="center" valign="bottom"
|style="font-weight:bold" height="15" | Noi
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" |
| align="center" |
| align="center" |
| align="center" |
| align="center" |
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | V4S3
| align="center" | 13.2
| align="center" | 100
| align="center" | 8.9
| align="center" | 67.2
| align="center" | 2.2
| align="center" | 16.4
| align="center" | 24.4
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | V6S3
| align="center" | 15.9
| align="center" | 100
| align="center" | 12.5
| align="center" | 78.4
| align="center" | 2.6
| align="center" | 16.1
| align="center" | 20.5
|}
[[Daniel:Notebook/HiResChrPaint|Back to calendar]]

Latest revision as of 23:25, 29 April 2013

Probe Prep Test (Started 4/22/2013)[edit]

Back to calendar

Ethanol Precipitation (Continued from 4/26/2013)[edit]

5. Took samples from -80 and centrifuged at 14000 rpm for 25 minutes at 4 C
6. Removed supernatant and added 750 uL 75% EtOH
7. Centrifugd at 14000 rpm for 15 minutes at 4 C
8. Removed most supernatant with pipette and then dried in hood
9. Resuspended in 8 uL nuclease free water

TBU gel Quantification[edit]

1. Preheated TBE buffer in 60 C incubator
2. microwaved for 1 minute
3. Added hot TBE to gel box and immediately preran gel for 20 minutes at 250V
4. Created samples according to following table
Lane 3 4 5 6 7 8 9 10
Sample Ladder Ladder V4S3 V4S3 V6S3 V6S3 Ladder Buffer
uL sample 0.5 1 0.5 0.5 0.5 0.5 0.5 0
uL hot TBE 5.5 5 5.5 5.5 5.5 5.5 5.5 6
uL 2x buffer 6 6 6 6 6 6 6 6
loaded 12 12 12 12 12 12 12 12
5. Incubated samples at 65C for 8 minutes
6. Cold snapped with ice box
7. Loaded 12 uL each sample and ran gel for 20 minutes at 250V
8. Stained with 3 uL SYBR gold for 25 minutes

Gel Results[edit]

Volume Report: File:GelQuant 042713.xls

File:GelQuant 042713.jpg

File:GelQuant analysis 042713.jpg

Sample Concentrations[edit]

Sample ng/uL ng (7 uL)
V4S3 4.6 32.4
V4S3 3.1 22.0
V6S3 3.5 24.7

Discussion[edit]

With the abysmally low yields from the Lambda Exonuclease reaction, I have come to the conclusions that it is this reaction causing the 
  most problems in my probe prep protocol.  I'm going to need to approach this reaction better before I continue.  The following table
  summarizes the yields in (ug and %) comparing my probe preps and Noi's:
  qPCR (ug) qPCR Yield (%) DpnII (ug) DpnII Yield (%) Lambda Exo (ug) Lambda Exo Yield (%) Yield from DPNII
Me              
V4S3 8.2 100 5.6 68.3 0.03 0.4 0.6
V6S3 7.4 100 4.7 63.5 0.02 0.3 0.5
Noi    
V4S3 13.2 100 8.9 67.2 2.2 16.4 24.4
V6S3 15.9 100 12.5 78.4 2.6 16.1 20.5

Back to calendar