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]

Back to Calendar

DpnII Digest (from 07/08/2014)[edit]

  1. Column Purification, elute with 40 uL
  2. 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

File:2014 07 09 DpnII.jpg

Lambda Exonuclease Digestion[edit]

  1. 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
  1. Incubate for 1 hr at 37C
  2. Heat inactivate for 10 minutes at 75C
  3. 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.