Daniel:Notebook/HiResChrPaint/2013-7-23: Difference between revisions
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>Djacobse (Created page with "=S2 Probe Preparation= Back to Calendar ==DpnII Digestion (from 7/18/2013)== 3. Pool samples ...") |
>Djacobse No edit summary |
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=S2 Probe Preparation= | =S2 Probe Preparation ([[Daniel:Notebook/HiResChrPaint/2013-7-22|Started 7/22/2013]])= | ||
[[Daniel:Notebook/HiResChrPaint|Back to Calendar]] | [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] | ||
==DpnII Digestion ([[Daniel:Notebook/HiResChrPaint/2013-7- | ==DpnII Digestion ([[Daniel:Notebook/HiResChrPaint/2013-7-22|from 7/22/2013]])== | ||
3. Pool samples for V4 set into 4AB and 4CD | 3. Pool samples for V4 set into 4AB and 4CD | ||
4. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 30 uL EB | 4. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 30 uL EB** | ||
5. Measure concentrations in nanodrop | 5. Pooled 4AB and 4CD into V4S2 and pooled V6 sets into V6AB and V6CD | ||
6. Measure concentrations in nanodrop | |||
Note (**): Forgot to move the columns to fresh tubes before elution. Caught myself just as the centrifuge started, so I stopped it immediately, before it even got to full speed. Still, this resulted in me losing most of the samples (I also threw away the collection tubes before proceeding to the fresh collection tube). | |||
===Nanodrop Results=== | ===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" | Sample | |||
| width="65" | V4S2 | |||
| width="65" | V6S2AB | |||
| width="65" | V6S2CD | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ng/uL | |||
| align="center" | 142.9 | |||
| align="center" | 32.8 | |||
| align="center" | 34.9 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ug in 20 uL | |||
| align="center" | 2.9 | |||
| align="center" | 0.7 | |||
| align="center" | 0.7 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | total ug | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | 1.4 | |||
|} | |||
===DpnII Digest gel=== | ===DpnII Digest gel=== | ||
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1. Lanes; (5.5 uL TBE; 2 uL 6x buffer, 0.5 uL sample; load 7 uL for all) | 1. Lanes; (5.5 uL TBE; 2 uL 6x buffer, 0.5 uL sample; load 7 uL for all) | ||
{{GelLanes| | {{GelLanes|Lane3=25bp ladder|Lane4=V4S2|Lane5=V6S2AB|Lane6=V4S2CD}} | ||
===Discussion=== | |||
Since I botched this probe prep, I may have to start over. I still have to digest with lambda exonuclease, which is going to drop my concentrations by 50% regardless. This would mean I'd likely have <1 ug for both V4S2 and V6S2 prior to dye coupling. It's just not worth it. |
Revision as of 19:11, 23 July 2013
S2 Probe Preparation (Started 7/22/2013)
DpnII Digestion (from 7/22/2013)
3. Pool samples for V4 set into 4AB and 4CD 4. Column Purification, elute with 30 uL EB** 5. Pooled 4AB and 4CD into V4S2 and pooled V6 sets into V6AB and V6CD 6. Measure concentrations in nanodrop
Note (**): Forgot to move the columns to fresh tubes before elution. Caught myself just as the centrifuge started, so I stopped it immediately, before it even got to full speed. Still, this resulted in me losing most of the samples (I also threw away the collection tubes before proceeding to the fresh collection tube).
Nanodrop Results
Sample | V4S2 | V6S2AB | V6S2CD |
ng/uL | 142.9 | 32.8 | 34.9 |
ug in 20 uL | 2.9 | 0.7 | 0.7 |
total ug | 1.4 |
DpnII Digest gel
1. Lanes; (5.5 uL TBE; 2 uL 6x buffer, 0.5 uL sample; load 7 uL for all)
Lane | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
Sample | 25bp ladder | V4S2 | V6S2AB | V4S2CD |
Discussion
Since I botched this probe prep, I may have to start over. I still have to digest with lambda exonuclease, which is going to drop my concentrations by 50% regardless. This would mean I'd likely have <1 ug for both V4S2 and V6S2 prior to dye coupling. It's just not worth it.