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

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[[Image:TBE_042913.jpg|600px]]
[[Image:TBE_042913.jpg|600px]]
==Lambda Exonuclease Digestion==
[[Daniel:Reagents/LambdaExonuclease| Enzyme Details]]
===Protocol===
1. Set up following reaction
  *Lambda Exo: NEB M0262L; Buffer: NEB B0262S
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:12pt" align="center"
| width="65" height="30" | Sample
| width="65" | ng/uL
| width="65" | uL
| width="65" | ng
| width="59" | Incubation Time
| width="65" | uL buffer
| width="65" | uL lambda exo (5U/uL)
| width="65" | uL nf H20
| width="65" | Total
|- style="font-size:12pt" align="center"
| height="15" | V6S2
| align="center" | 27.3
| align="center" | 25
| align="center" | 682.5
| 1 hr
| align="center" | 5
| align="center" | 0.5
| align="center" | 19.5
| align="center" | 50
|- style="font-size:12pt" align="center"
| height="15" | V6S3
| align="center" | 27.6
| align="center" | 25
| align="center" | 690
| 1 hr
| align="center" | 5
| align="center" | 2
| align="center" | 18
| align="center" | 50
|- style="font-size:12pt" align="center"
| height="15" | V6S4
| align="center" | 25.3
| align="center" | 25
| align="center" | 632.5
| 30 min
| align="center" | 5
| align="center" | 2
| align="center" | 18
| align="center" | 50
|- style="font-size:12pt" align="center"
| height="15" | V4S2
| align="center" | 37
| align="center" | 25
| align="center" | 925
| 2 hr
| align="center" | 5
| align="center" | 2
| align="center" | 18
| align="center" | 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==

Revision as of 23:13, 29 April 2013

Probe Prep Test (Started 4/22/2013)

Back to calendar

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

Protocol

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