Daniel:Notebook/HiResChrPaint/2013-9-4: Difference between revisions
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>Djacobse (Created page with "=BSPP Control (Started 09/03/2013)= Back to Calendar ==DpnII Digestion ([[Daniel:Notebook/HiResC...") |
>Djacobse |
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(9 intermediate revisions by the same user not shown) | |||
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===Nanodrop Results=== | ===Nanodrop Results=== | ||
===Gel | {| 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" | BSPP 1AB | |||
| width="65" | BSPP 1CD | |||
| width="65" | BSPP 2AB | |||
| width="65" | BSPP 2CD | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ng/uL | |||
| align="center" | 127.7 | |||
| align="center" | 130.6 | |||
| align="center" | 110.1 | |||
| align="center" | 109.3 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ug in 30 uL | |||
| align="center" | 3.8 | |||
| align="center" | 3.9 | |||
| align="center" | 3.3 | |||
| align="center" | 3.3 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | total | |||
| align="center" | | |||
| align="center" | 7.7 | |||
| align="center" | | |||
| align="center" | 6.6 | |||
|} | |||
===TBE Gel=== | |||
1. Lanes; (5.5 uL TBE; 2 uL 6x buffer, 0.5 uL sample; load 7 uL for all) | |||
{{GelLanes|Lane4=25bp ladder|Lane5=BSPP 1AB|Lane6=BSPP 1CD|Lane7=BSPP 2AB|Lane8=BSPP 2CD}} | |||
2. Run for 30 minutes at 200V | |||
3. Stain with 3 uL SYBR gold for 2 minutes | |||
'''Gel Image''' | |||
[[Image:2013-09-04-DpnIIDigest-BSPP.jpg|600px]] | |||
The gel looks fine, so I'm going to proceed as usual. | |||
==Lambda Exonuclease Digestion== | ==Lambda Exonuclease Digestion== | ||
1. Reaction table | |||
{| 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" | BSPP 1AB | |||
| width="65" | BSPP 1CD | |||
| width="65" | BSPP 2AB | |||
| width="65" | BSPP 2CD | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ng/uL | |||
| align="center" | 127.7 | |||
| align="center" | 130.6 | |||
| align="center" | 110.1 | |||
| align="center" | 109.3 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ug in 25 uL | |||
| align="center" | 3.2 | |||
| align="center" | 3.3 | |||
| align="center" | 2.8 | |||
| align="center" | 2.7 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | uL added | |||
| align="center" | 25 | |||
| align="center" | 25 | |||
| align="center" | 25 | |||
| align="center" | 25 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="30" | uL 10x buffer | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="45" | uL Lambda Exonuclease (5U/uL) | |||
| align="center" | 3 | |||
| align="center" | 3 | |||
| align="center" | 3 | |||
| align="center" | 3 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | uL nfH2O | |||
| align="center" | 17 | |||
| align="center" | 17 | |||
| align="center" | 17 | |||
| align="center" | 17 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | total | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
|} | |||
2. Incubate for 1 hour at 37C followed by 10 minute heat inactivation at 75C | |||
3. Pool samples | |||
4. [[Daniel:Protocols/ssDNA Column|ssDNA column]] purification, elute with 20 uL | |||
5. Measure Concentration in Nanodrop | |||
===ssNanodrop 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" | ssProbe | |||
| width="65" | BSPP 1 | |||
| width="65" | BSPP 2 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ng/uL | |||
| align="center" | 119 | |||
| align="center" | 100.3 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ug in 20 uL | |||
| align="center" | 2.4 | |||
| align="center" | 2.0 | |||
|} |
Latest revision as of 00:15, 5 September 2013
BSPP Control (Started 09/03/2013)[edit]
DpnII Digestion (Started 09/03/2013)[edit]
3. Column Purification, elute with 30 uL EB 4. Measure concentrations in nanodrop
Nanodrop Results[edit]
Sample | BSPP 1AB | BSPP 1CD | BSPP 2AB | BSPP 2CD |
ng/uL | 127.7 | 130.6 | 110.1 | 109.3 |
ug in 30 uL | 3.8 | 3.9 | 3.3 | 3.3 |
total | 7.7 | 6.6 |
TBE Gel[edit]
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 | BSPP 1AB | BSPP 1CD | BSPP 2AB | BSPP 2CD |
2. Run for 30 minutes at 200V 3. Stain with 3 uL SYBR gold for 2 minutes
Gel Image
File:2013-09-04-DpnIIDigest-BSPP.jpg
The gel looks fine, so I'm going to proceed as usual.
Lambda Exonuclease Digestion[edit]
1. Reaction table
Sample | BSPP 1AB | BSPP 1CD | BSPP 2AB | BSPP 2CD |
ng/uL | 127.7 | 130.6 | 110.1 | 109.3 |
ug in 25 uL | 3.2 | 3.3 | 2.8 | 2.7 |
uL added | 25 | 25 | 25 | 25 |
uL 10x buffer | 5 | 5 | 5 | 5 |
uL Lambda Exonuclease (5U/uL) | 3 | 3 | 3 | 3 |
uL nfH2O | 17 | 17 | 17 | 17 |
total | 50 | 50 | 50 | 50 |
2. Incubate for 1 hour at 37C followed by 10 minute heat inactivation at 75C 3. Pool samples 4. ssDNA column purification, elute with 20 uL 5. Measure Concentration in Nanodrop
ssNanodrop Results[edit]
ssProbe | BSPP 1 | BSPP 2 |
ng/uL | 119 | 100.3 |
ug in 20 uL | 2.4 | 2.0 |