Daniel:Notebook/HiResChrPaint/2013-7-17

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S3 Probe Preparation (Started 7/16/2013)[edit]

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DpnII Digestion[edit]

3. Column Purification, elute with 30 uL EB
4. Measure concentrations in nanodrop

Nanodrop Results[edit]

Sample V4S3A V4S3B V4S3C V4S3D V6S3A V6S3B V6S3C V6S3D
ng/uL 84.9 82.3 85.4 86.4 107.6 98.2 107.4 109.2
ug in 30 uL 2.5 2.5 2.6 2.6 3.2 2.9 3.2 3.3
total ug       10.2       12.7

DpnII Digest 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 10bp ladder V4S3A V4S3B V4S3C V4S3D V6S3A V6S3B V6S3C V6S3D
2. Run for 25 minutes at 250V

Gel Image

File:DpnIIDigest 071713.jpg

Lambda Exonuclease Digestion[edit]

1. Reaction table
Sample V4S3A V4S3B V4S3C V4S3D V6S3A V6S3B V6S3C V6S3D
ng/uL 84.9 82.3 85.4 86.4 107.6 98.2 107.4 109.2
ug in 25 uL 2.1 2.1 2.1 2.2 2.7 2.5 2.7 2.7
uL added 25 25 25 25 25 25 25 25
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 nfH2O 17 17 17 17 17 17 17 17
total 50 50 50 50 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 (different from before)
6. Dry samples in vaccuum centrifuge

Nanodrop Results[edit]

Sample V4S3 V6S3
ng/uL 166 228.1
ug in 20 uL 3.3 4.6

Dye Coupling[edit]

For each

1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
2. Resuspend DNA in 5 uL nfH20
3. Add 5 uL DNA and 3 uL of sodium bicarbonate buffer in PCR tube
4. Denature samples for 5 minutes at 95C, then snap cool using ice box
5. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
   *dye cannot be saved for later use.  Use immediately!
6. Add 2 uL dye to sample
7. Incubate in the dark for 1 hour
8. Add 10 uL nfH20 to sample
9. Purify using Centri Sep column
10. Measure concentrations using Microarray setting in nanodrop

Labeling Results[edit]

  ng/uL DNA pmol/uL dye ug DNA in 15 uL pmol/uL DNA dye/probe dye/100 bp Base:Dye Ratio (:1)
V4S3 140.3 7.5 2.1 5.2 1.4 1.8 57
V6S3 142.9 7.5 2.1 5.3 1.4 1.7 58

Discussion on S3 Probe Preparation[edit]

This probe prep took me two days. By the end I have approximately 2 ug of DNA per sample, with labeling ratios of around 50:1 bp:dye.