Daniel:Notebook/HiResChrPaint/2014-7-9: Difference between revisions
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Scary that both of the columns yielded the same percent (I double checked the math). As a note, taking into account that the theoretical maximum yield is 80% (since we remove 20bp out of 100 on each strand), this is approximately a 90% yield from the column. | Scary that both of the columns yielded the same percent (I double checked the math). As a note, taking into account that the theoretical maximum yield is 80% (since we remove 20bp out of 100 on each strand), this is approximately a '''90% yield''' from the column. | ||
===TBE Gel=== | |||
'''Gel Image''' | |||
[[Image:2014_07_09_DpnII.jpg|500px]] | |||
==Lambda Exonuclease Digestion== | ==Lambda Exonuclease Digestion== | ||
Line 54: | Line 60: | ||
#Reaction Table | #Reaction Table | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#C5D9F1;font-size:12pt;font-weight:bold" | |||
| width="119" height="15" valign="bottom" | Sample | |||
| width="65" align="center" valign="bottom" | V6S2AB | |||
| width="65" align="center" valign="bottom" | V6S2CD | |||
| width="65" align="center" valign="bottom" | V6S3AB | |||
| width="65" align="center" valign="bottom" | V6S3CD | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | ng/uL | |||
| align="center" align="center" valign="bottom" | 122.4 | |||
| align="center" align="center" valign="bottom" | 110.7 | |||
| align="center" align="center" valign="bottom" | 137 | |||
| align="center" align="center" valign="bottom" | 133.5 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | ug in 35 uL | |||
| align="center" align="center" valign="bottom" | 4.3 | |||
| align="center" align="center" valign="bottom" | 3.9 | |||
| align="center" align="center" valign="bottom" | 4.8 | |||
| align="center" align="center" valign="bottom" | 4.7 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | uL sample | |||
| align="center" align="center" valign="bottom" | 35 | |||
| align="center" align="center" valign="bottom" | 35 | |||
| align="center" align="center" valign="bottom" | 35 | |||
| align="center" align="center" valign="bottom" | 35 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | uL 10X buffer | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="30" valign="bottom" | uL Lambda Exonuclease (5 U/uL) | |||
| align="center" align="center" valign="bottom" | 3 | |||
| align="center" align="center" valign="bottom" | 3 | |||
| align="center" align="center" valign="bottom" | 3 | |||
| align="center" align="center" valign="bottom" | 3 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | uL nfH2O | |||
| align="center" align="center" valign="bottom" | 7 | |||
| align="center" align="center" valign="bottom" | 7 | |||
| align="center" align="center" valign="bottom" | 7 | |||
| align="center" align="center" valign="bottom" | 7 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Total | |||
| align="center" align="center" valign="bottom" | 50 | |||
| align="center" align="center" valign="bottom" | 50 | |||
| align="center" align="center" valign="bottom" | 50 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|} | |||
#Incubate for 1 hr at 37C | #Incubate for 1 hr at 37C | ||
#Heat inactivate for 10 minutes at 75C | #Heat inactivate for 10 minutes at 75C | ||
#Purify with [[Daniel:Protocols/ssDNA Column|Zymo column]], elute with 20 uL nfH2O | #Purify with [[Daniel:Protocols/ssDNA Column|Zymo column]], elute with 20 uL nfH2O | ||
===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" | |||
| width="82" height="15" valign="bottom" | Sample | |||
| width="65" align="center" valign="bottom" | V6S2 | |||
| width="65" align="center" valign="bottom" | V6S3 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | ng/uL ssDNA | |||
| align="center" align="center" valign="bottom" | 187.6 | |||
| align="center" align="center" valign="bottom" | 228.1 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | ug ssDNA | |||
| align="center" align="center" valign="bottom" | 3.8 | |||
| align="center" align="center" valign="bottom" | 4.6 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="30" valign="bottom" | Yield (Max ~50%) | |||
| align="center" align="center" valign="bottom" | 42.16 | |||
| align="center" align="center" valign="bottom" | 48.02 | |||
|} | |||
Again, since the theoretical maximum of this reaction is 50% (we eliminate 1 strand), this yields an effective 85% and 96% yield from the reaction/column, which is pretty good. | |||
[[Category:HRCP]] [[Category:20140708]] [[Category:Lambda Exo]] |
Latest revision as of 21:42, 9 July 2014
Strand Control (Started 07/08/2014)[edit]
DpnII Digest (from 07/08/2014)[edit]
- Column Purification, elute with 40 uL
- Measure concentration in nanodrop
Nanodrop Results[edit]
Sample | V6S2AB | V6S2CD | V6S3AB | V6S3CD |
ng/uL | 122.4 | 110.7 | 137 | 133.5 |
ug in 40 uL | 4.9 | 4.4 | 5.5 | 5.3 |
ug total | 9.3 | 10.8 | ||
Yield (Max ~80%) | 71.18 | 71.18 |
Scary that both of the columns yielded the same percent (I double checked the math). As a note, taking into account that the theoretical maximum yield is 80% (since we remove 20bp out of 100 on each strand), this is approximately a 90% yield from the column.
TBE Gel[edit]
Gel Image
Lambda Exonuclease Digestion[edit]
- Reaction Table
Sample | V6S2AB | V6S2CD | V6S3AB | V6S3CD |
ng/uL | 122.4 | 110.7 | 137 | 133.5 |
ug in 35 uL | 4.3 | 3.9 | 4.8 | 4.7 |
uL sample | 35 | 35 | 35 | 35 |
uL 10X buffer | 5 | 5 | 5 | 5 |
uL Lambda Exonuclease (5 U/uL) | 3 | 3 | 3 | 3 |
uL nfH2O | 7 | 7 | 7 | 7 |
Total | 50 | 50 | 50 | 50 |
- Incubate for 1 hr at 37C
- Heat inactivate for 10 minutes at 75C
- Purify with Zymo column, elute with 20 uL nfH2O
Nanodrop Results[edit]
Sample | V6S2 | V6S3 |
ng/uL ssDNA | 187.6 | 228.1 |
ug ssDNA | 3.8 | 4.6 |
Yield (Max ~50%) | 42.16 | 48.02 |
Again, since the theoretical maximum of this reaction is 50% (we eliminate 1 strand), this yields an effective 85% and 96% yield from the reaction/column, which is pretty good.