Daniel:Notebook/HiResChrPaint/2013-4-29: Difference between revisions
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==Ethanol Precipitation== | ==Ethanol Precipitation== | ||
[[Daniel:Protocols/EtOHPrecip| Standard Protocol]] | |||
1. Added 250 uL nuclease free water to each reaction | 1. Added 250 uL nuclease free water to each reaction | ||
2. | 2. Added 900 uL 100% EtOH, 30 uL NaOAc, 1 uL glycoblue | ||
3. Incubate overnight at -80C | |||
4. Continued [[Daniel:Notebook/HiResChrPaint/2013-4-30|4/30/2013]] |
Latest revision as of 23:17, 29 April 2013
Probe Prep Test (Started 4/22/2013)[edit]
TBE Gel[edit]
Purpose: As per 4/27/2013 results, I need to better understand the lambda exonuclease digestion. To this end I'm going to play around with the parameters of the reaction. To do so I need several samples to work with. I am testing/identifying samples right now that will suit my purposes.
Protocol[edit]
1. Prepared samples (1 uL sample, 5 uL TBE, 2 uL 6x dye) and loaded (7 uL) according to table
Lane | 2 | 3 | 4 | 5 | 6 |
Sample | Ladder | V4S2 | V6S2 | V6S3 | V6S3 Digest |
2. Ran gel for 25 minutes at 200 V 3. Stained with 3 uL SYBR gold for 5 min
Gel Results[edit]
Lambda Exonuclease Digestion[edit]
1. Set up following reaction *Lambda Exo: NEB M0262L; Buffer: NEB B0262S
Sample | ng/uL | uL | ng | Incubation Time | uL buffer | uL lambda exo (5U/uL) | uL nf H20 | Total |
V6S2 | 27.3 | 25 | 682.5 | 1 hr | 5 | 0.5 | 19.5 | 50 |
V6S3 | 27.6 | 25 | 690 | 1 hr | 5 | 2 | 18 | 50 |
V6S4 | 25.3 | 25 | 632.5 | 30 min | 5 | 2 | 18 | 50 |
V4S2 | 37 | 25 | 925 | 2 hr | 5 | 2 | 18 | 50 |
3. Incubated in 37C incubator; heat shocked for 20 minutes at 65 C in thermocycler 4. Immediately proceeded to EtOH precipitation with every sample
Ethanol Precipitation[edit]
1. Added 250 uL nuclease free water to each reaction 2. Added 900 uL 100% EtOH, 30 uL NaOAc, 1 uL glycoblue 3. Incubate overnight at -80C 4. Continued 4/30/2013