Daniel:Notebook/HiResChrPaint/2013-5-13: Difference between revisions

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  1. 0.5 uL sample; 6 uL TBE, 2 uL dye per sample
  1. 0.5 uL sample; 6 uL TBE, 2 uL dye per sample
  2. Gel Arrangement:
  2. Gel Arrangement(5-12 are digested samples that have been column purified, ready to use for lambda exo digestion):


{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
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===Gel Results===
===Gel Results===
[[Image:DpnDigest_051313.jpg|600px]]
==Lambda Exonuclease Digestion==
Since I still have plenty of DNA, I'm continuing with the lambda exonuclease digestion.  At the end I will be pooling the samples and running
them on the ssDNA column.
1. Prepared samples according to 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="85" height="15" | Sample
| width="65" | V4S3A
| width="65" | V4S3B
| width="65" | V4S3C
| width="65" | V4S3D
| width="65" | V6S3A
| width="65" | V6S3B
| width="65" | V6S3C
| width="65" | V6S3D
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ng/uL
| align="center" | 115.1
| align="center" | 138.1
| align="center" | 120.3
| align="center" | 124.4
| align="center" | 138.9
| align="center" | 131.4
| align="center" | 133.5
| align="center" | 127.3
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | uL sample
| align="center" | 25
| align="center" | 25
| align="center" | 25
| align="center" | 25
| align="center" | 25
| align="center" | 25
| align="center" | 25
| align="center" | 25
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ug sample
| align="center" | 2.9
| align="center" | 3.5
| align="center" | 3.0
| align="center" | 3.1
| align="center" | 3.5
| align="center" | 3.3
| align="center" | 3.3
| align="center" | 3.2
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="27" | uL 10X buffer
| align="center" | 5
| align="center" | 5
| align="center" | 5
| align="center" | 5
| 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
| 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 nf H20
| align="center" | 17
| align="center" | 17
| align="center" | 17
| align="center" | 17
| 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
| align="center" | 50
| align="center" | 50
| align="center" | 50
| align="center" | 50
|}
2. Incubate 1 hour at 37C followed by 10 minutes at 75C
3. Pool samples (4x50 -> 1x200 uL)
4. ssColumn Purification
==TBU Gel Quantification==
[[Daniel:Protocols/TBUgel|Protocol 2]]
1. Prepared samples according to 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" | Lane
| align="center" width="65" | 2
| align="center" width="65" | 3
| align="center" width="65" | 4
| align="center" width="65" | 5
| align="center" width="65" | 6
| align="center" width="65" | 7
| align="center" width="65" | 10
| align="center" width="65" | 11
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="45" | Sample
| Ladder
| V4S3A Undigested
| V6S3D Undigested
| V4S3
| V6S3
| Ladder
| V4S3C Pre-Column
| V6S3C Pre-Column
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | uL sample
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 0.5
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="27" | uL TBE
| align="center" | 5.5
| align="center" | 5.5
| align="center" | 5.5
| align="center" | 5.5
| align="center" | 5.5
| align="center" | 5.5
| align="center" | 5.5
| align="center" | 5.5
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="45" | uL 2X Urea Buffer
| align="center" | 6
| align="center" | 6
| align="center" | 6
| align="center" | 6
| align="center" | 6
| align="center" | 6
| align="center" | 6
| align="center" | 6
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | Loaded
| align="center" | 4
| align="center" | 4
| align="center" | 4
| align="center" | 4
| align="center" | 4
| align="center" | 4
| align="center" | 4
| align="center" | 4
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | Dilution
| align="center" | 0.1
| align="center" | 0.1
| align="center" | 0.1
| align="center" | 0.1
| align="center" | 0.1
| align="center" | 0.1
| align="center" | 0.1
| align="center" | 0.1
|}
2. Run for 25 minutes at 250V
3. Incubate for 20 minutes with 3 uL SYBR gold
===Gel Results===
[[GelQuant_051313.jpg|600px]]

Latest revision as of 00:03, 14 May 2013

Probe Prep Test 2 (Started 5/10/2013)[edit]

Back to Calendar

TBE Gel[edit]

1. 0.5 uL sample; 6 uL TBE, 2 uL dye per sample
2. Gel Arrangement(5-12 are digested samples that have been column purified, ready to use for lambda exo digestion):
Lane 1 2 3 4 5 6 7 8 9 10 11 12
Sample 25bp Ladder V6S3A Undigested V4S3C Pre-Column V6S3B Pre-Column V4S3A V4S3B V4S3C V4S3D V6S3A V6S3B V6S3C V6S3D
3. Loaded 7 uL per sample
4. Ran gel for 30 minutes at 200V
5. Stained 2 minutes with 3 uL SYBR gold

Gel Results[edit]

File:DpnDigest 051313.jpg

Lambda Exonuclease Digestion[edit]

Since I still have plenty of DNA, I'm continuing with the lambda exonuclease digestion.  At the end I will be pooling the samples and running 
them on the ssDNA column.
1. Prepared samples according to table:
Sample V4S3A V4S3B V4S3C V4S3D V6S3A V6S3B V6S3C V6S3D
ng/uL 115.1 138.1 120.3 124.4 138.9 131.4 133.5 127.3
uL sample 25 25 25 25 25 25 25 25
ug sample 2.9 3.5 3.0 3.1 3.5 3.3 3.3 3.2
uL 10X buffer 5 5 5 5 5 5 5 5
uL Lambda Exonuclease (5U/uL) 3 3 3 3 3 3 3 3
uL nf H20 17 17 17 17 17 17 17 17
Total 50 50 50 50 50 50 50 50
2. Incubate 1 hour at 37C followed by 10 minutes at 75C
3. Pool samples (4x50 -> 1x200 uL)
4. ssColumn Purification

TBU Gel Quantification[edit]

Protocol 2

1. Prepared samples according to table
Lane 2 3 4 5 6 7 10 11
Sample Ladder V4S3A Undigested V6S3D Undigested V4S3 V6S3 Ladder V4S3C Pre-Column V6S3C Pre-Column
uL sample 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
uL TBE 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5
uL 2X Urea Buffer 6 6 6 6 6 6 6 6
Loaded 4 4 4 4 4 4 4 4
Dilution 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
2. Run for 25 minutes at 250V
3. Incubate for 20 minutes with 3 uL SYBR gold

Gel Results[edit]

600px