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

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(Autocreate 2014/04/29 Entry for Haynes_Lab:Notebook/Characterizing_AHL_quorum_sensing_homologs)
 
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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.  
<br>
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).
<br>
Controls
Positive control was a miniprepped plasmid at 100ng/ul concentration.
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Volume (ul)'''
| align="center" style="background:#f0f0f0;"|''''''
|-
| Thing||Positive ctrl||Negative ctrl
|-
| H2O||12||12
|-
| Green digest buffer||2||2
|-
| pTet mCh||5||5
|-
| DpnI||1||0
|}


<br><br>


{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Thing'''
| align="center" style="background:#f0f0f0;"|'''Volume (ul)'''
|-
| 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.<br>
Cleaned using zymo DNA clean and concentration-5 kit (columns from gel extraction kit). Added 150ul of DNA binding buffer to DpnI reactions.
<br>
Gel extracted PCR reaction 1. Saw a smaller band above, think I got the correct band.
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Revision as of 13:03, 29 April 2014

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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 Volume (ul)
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.


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