Daniel:Notebook/HiResChrPaint/2013-5-13
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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