Daniel:Notebook/HiResChrPaint/2013-4-29: Difference between revisions

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[[Daniel:Reagents/LambdaExonuclease| Enzyme Details]]
[[Daniel:Reagents/LambdaExonuclease| Enzyme Details]]
===Protocol===


  1. Set up following reaction
  1. Set up following reaction
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==Ethanol Precipitation==
==Ethanol Precipitation==
[[Daniel:Protocols/EtOHPrecip| Standard Protocol]]
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 [[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]

Back to calendar

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]

File:TBE 042913.jpg

Lambda Exonuclease Digestion[edit]

Enzyme Details

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]

Standard Protocol

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