Revision as of 23:17, 29 April 2013 by >Djacobse
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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
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