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 | ||
Line 13: | Line 13: | ||
==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]
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...