Revision as of 18:29, 13 May 2013 by >Djacobse
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TBE Gel
1. 0.5 uL sample; 6 uL TBE, 2 uL dye per sample
2. Gel Arrangement(5-12 are digested samples that have been column purified, ready to use for lambda exo digestion):
Lane
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1
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2
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3
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4
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5
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6
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7
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8
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9
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10
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11
|
12
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Sample
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25bp Ladder
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V6S3A Undigested
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V4S3C Pre-Column
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V6S3B Pre-Column
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V4S3A
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V4S3B
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V4S3C
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V4S3D
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V6S3A
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V6S3B
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V6S3C
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V6S3D
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3. Loaded 7 uL per sample
4. Ran gel for 30 minutes at 200V
5. Stained 2 minutes with 3 uL SYBR gold
Gel Results
File:DpnDigest 051313.jpg
Lambda Exonuclease Digestion
Since I still have plenty of DNA, I'm continuing with the lambda exonuclease digestion. At the end I will be pooling the samples and running
them on the ssDNA column.
1. Prepared samples according to table:
Sample
|
V4S3A
|
V4S3B
|
V4S3C
|
V4S3D
|
V6S3A
|
V6S3B
|
V6S3C
|
V6S3D
|
ng/uL
|
115.1
|
138.1
|
120.3
|
124.4
|
138.9
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131.4
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133.5
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127.3
|
uL sample
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25
|
25
|
25
|
25
|
25
|
25
|
25
|
25
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ug sample
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2.9
|
3.5
|
3.0
|
3.1
|
3.5
|
3.3
|
3.3
|
3.2
|
uL 10X buffer
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
uL Lambda Exonuclease (5U/uL)
|
3
|
3
|
3
|
3
|
3
|
3
|
3
|
3
|
uL nf H20
|
17
|
17
|
17
|
17
|
17
|
17
|
17
|
17
|
Total
|
50
|
50
|
50
|
50
|
50
|
50
|
50
|
50
|
2. Incubate 1 hour at 37C followed by 10 minutes at 75C
3. Pool samples (4x50 -> 1x200 uL)
4. ssColumn Purification