Daniel:Notebook/HiResChrPaint/2013-5-13: Difference between revisions
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>Djacobse (Created page with "=Probe Prep Test 2 (Started 5/10/2013)= Back to Calendar ==TBE Gel== 1. 0.5 uL sample; 6 uL T...") |
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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> | ||
Line 46: | Line 46: | ||
===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]
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]
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]
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