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mm/dd/yyyy
Ran PCR products from yesterday on a gel. 5ul of reactions 2-9, all 30 of 1 so I can gel extract because there should be two bands and I only want one.
DpnI digest PCR reactions 2-7 and 9 with positive and negative controls (didn't run reaction 8 because I checked the gel early and saw that there was no DNA there).
Controls
Positive control was a miniprepped plasmid at 100ng/ul concentration.
'
|
Volume (ul)
|
'
|
Thing |
Positive ctrl |
Negative ctrl
|
H2O |
12 |
12
|
Green digest buffer |
2 |
2
|
pTet mCh |
5 |
5
|
DpnI |
1 |
0
|
Thing
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Volume (ul)
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Digest buffer |
4
|
PCR rxn |
25
|
H2O |
1
|
DpnI |
1
|
Incubated at 37°C for 55 minutes, sat on the bench for 3 minutes, heat inactivated at 80 for 5 minutes.
Cleaned using zymo DNA clean and concentration-5 kit (columns from gel extraction kit). Added 150ul of DNA binding buffer to DpnI reactions.
Gel extracted PCR reaction 1. Saw a smaller band above, think I got the correct band.
PCR reaction
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Description
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'
|
260
|
280
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260/280
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ng/µL
|
1 |
Rec bb |
Gel extracted |
0.02 |
0.01 |
2 |
20.289
|
2 |
pLux-GFP |
DpnI digested, column purified |
0.047 |
0.024 |
2 |
47.148
|
3 |
pRhl-GFP |
DpnI digested, column purified |
0.05 |
0.025 |
2.004 |
50.466
|
4 |
pEsa-GFP |
DpnI digested, column purified |
0.032 |
0.015 |
2.117 |
32.1
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5 |
pLac-LuxR |
DpnI digested, column purified |
0.046 |
0.024 |
1.935 |
45.771
|
6 |
pLac-RhlR |
DpnI digested, column purified |
0.06 |
0.035 |
1.692 |
59.88
|
7 |
pLac-RhlR |
DpnI digested, column purified |
0.041 |
0.019 |
2.2 |
41.438
|
9 |
pLac-EsaR |
DpnI digested, column purified |
0.038 |
0.018 |
2.135 |
37.847
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