Daniel:Notebook/HiResChrPaint/2013-5-13

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

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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]

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