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
 
(9 intermediate revisions by the same user not shown)
Line 10: Line 10:
===Nanodrop Results===
===Nanodrop Results===


===Gel Image===
{| 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" | &nbsp;
| align="center" | 7.7
| align="center" | &nbsp;
| 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]

Back to Calendar

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