Daniel:Notebook/HiResChrPaint/2013-4-29

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Revision as of 23:14, 29 April 2013 by >Djacobse
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Probe Prep Test (Started 4/22/2013)

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TBE Gel

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

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

File:TBE 042913.jpg

Lambda Exonuclease Digestion

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

1. Added 250 uL nuclease free water to each reaction
2.