Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2014/04/29

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Current revision (15:45, 29 April 2014) (view source)
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Gel extracted PCR reaction 1. Saw a smaller band above, think I got the correct band.
Gel extracted PCR reaction 1. Saw a smaller band above, think I got the correct band.
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''PCR  reaction'''
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| align="center" style="background:#f0f0f0;"|'''Description'''
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| align="center" style="background:#f0f0f0;"|''''''
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| align="center" style="background:#f0f0f0;"|'''260'''
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| align="center" style="background:#f0f0f0;"|'''280'''
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| align="center" style="background:#f0f0f0;"|'''260/280'''
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| align="center" style="background:#f0f0f0;"|'''ng/µL'''
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|-
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| 1||Rec bb||Gel extracted||0.02||0.01||2||20.289
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| 2||pLux-GFP||DpnI digested, column purified||0.047||0.024||2||47.148
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|-
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| 3||pRhl-GFP||DpnI digested, column purified||0.05||0.025||2.004||50.466
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|-
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| 4||pEsa-GFP||DpnI digested, column purified||0.032||0.015||2.117||32.1
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|-
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| 5||pLac-LuxR||DpnI digested, column purified||0.046||0.024||1.935||45.771
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|-
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| 6||pLac-RhlR||DpnI digested, column purified||0.06||0.035||1.692||59.88
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| 7||pLac-RhlR||DpnI digested, column purified||0.041||0.019||2.2||41.438
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| 9||pLac-EsaR||DpnI digested, column purified||0.038||0.018||2.135||37.847
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|}
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Didn't have enough of the backbone so started another PCR. 1-4 are int receiver vector P112 P123, 5-6 are purified PCR product of receiver backbone P112 P123.
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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) '
ThingPositive ctrlNegative ctrl
H2O1212
Green digest buffer22
pTet mCh55
DpnI10



Thing Volume (ul)
Digest buffer4
PCR rxn25
H2O1
DpnI1

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 Description ' 260 280 260/280 ng/µL
1Rec bbGel extracted0.020.01220.289
2pLux-GFPDpnI digested, column purified0.0470.024247.148
3pRhl-GFPDpnI digested, column purified0.050.0252.00450.466
4pEsa-GFPDpnI digested, column purified0.0320.0152.11732.1
5pLac-LuxRDpnI digested, column purified0.0460.0241.93545.771
6pLac-RhlRDpnI digested, column purified0.060.0351.69259.88
7pLac-RhlRDpnI digested, column purified0.0410.0192.241.438
9pLac-EsaRDpnI digested, column purified0.0380.0182.13537.847

Didn't have enough of the backbone so started another PCR. 1-4 are int receiver vector P112 P123, 5-6 are purified PCR product of receiver backbone P112 P123.


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