Daniel:Notebook/HiResChrPaint/2013-5-4: 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/2013-5-3|5/3/2013]]==
==Ethanol Precipitation (Continued from [[Daniel:Notebook/HiResChrPaint/2013-5-3|5/3/2013]])==


  3. Spin at 12000 rpm for 30 minutes at 4 C
  3. Spin at 12000 rpm for 30 minutes at 4 C
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==TBU gel==
==TBU gel==


1. Sample table
  *Pre refers to before the Ethanol precipitation, post refers to after;
  **Two gels were run, one for V4S2, one for V6S2


{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:12pt" align="center"
| width="65" height="21" | Sample
| width="65" | Ladder
| width="65" | Ladder
| width="65" | Undigested
| width="65" | 30 min Pre
| width="65" | 30 min Post
| width="65" | 1 hr Pre
| width="65" | 1 hr Post
| width="65" | 2 hr Pre
| width="65" | 2 hr Post
|- style="font-size:12pt" align="center"
| height="15" | uL sample
| align="center" | 0.5
| align="center" | 1
| align="center" | 0.5
| align="center" | 1
| align="center" | 1
| align="center" | 1
| align="center" | 1
| align="center" | 1
| align="center" | 1
|- style="font-size:12pt" align="center"
| height="15" | uL TBE
| align="center" | 5.5
| align="center" | 5
| align="center" | 5.5
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 5
|- style="font-size:12pt" align="center"
| height="15" | uL 2X buffer
| align="center" | 6
| align="center" | 6
| align="center" | 6
| align="center" | 6
| align="center" | 6
| align="center" | 6
| align="center" | 6
| align="center" | 6
| align="center" | 6
|- style="font-size:12pt" align="center"
| height="15" | Loaded
| align="center" | 12
| align="center" | 12
| align="center" | 12
| align="center" | 12
| align="center" | 12
| align="center" | 12
| align="center" | 12
| align="center" | 12
| align="center" | 12
|}


A
A
Line 20: Line 85:
B
B
[[Image:LamdaDigest_050413B.jpg|600px]]
[[Image:LamdaDigest_050413B.jpg|600px]]
===Discussion===
Looking at the bands qualitatively, I would say there is no significant difference between 30 min, 1 hr, and 2 hr incubation times.
However, it does appear that quite a bit of DNA is lost in the Ethanol precipitation.  On [[Daniel:Notebook/HiResChrPaint/2013-5-6|Monday]] I'm going to test the Ethanol precipitation
against the ssDNA column prep that I bought previously.  Before when I used the column prep it didn't seem to be much better than the EtOH,
but we'll see...

Latest revision as of 17:16, 7 May 2013

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

Back to Calendar

Ethanol Precipitation (Continued from 5/3/2013)[edit]

3. Spin at 12000 rpm for 30 minutes at 4 C
4. Remove supernatant and add 750 uL chilled 75% EtOH
5. Centrifuge at 12000 rpm for 15 minutes at 4 C
6. Remove supernatant and dry in hood
7. Resuspend in 7 uL nuclease free H20

TBU gel[edit]

1. Sample table
 *Pre refers to before the Ethanol precipitation, post refers to after;
 **Two gels were run, one for V4S2, one for V6S2
Sample Ladder Ladder Undigested 30 min Pre 30 min Post 1 hr Pre 1 hr Post 2 hr Pre 2 hr Post
uL sample 0.5 1 0.5 1 1 1 1 1 1
uL TBE 5.5 5 5.5 5 5 5 5 5 5
uL 2X buffer 6 6 6 6 6 6 6 6 6
Loaded 12 12 12 12 12 12 12 12 12

A File:LamdaDigest 050413A.jpg

B File:LamdaDigest 050413B.jpg

Discussion[edit]

Looking at the bands qualitatively, I would say there is no significant difference between 30 min, 1 hr, and 2 hr incubation times.
However, it does appear that quite a bit of DNA is lost in the Ethanol precipitation.  On Monday I'm going to test the Ethanol precipitation
against the ssDNA column prep that I bought previously.  Before when I used the column prep it didn't seem to be much better than the EtOH,
but we'll see...