Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2014/04/07: Difference between revisions

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(Autocreate 2014/04/07 Entry for Haynes_Lab:Notebook/Characterizing_AHL_quorum_sensing_homologs)
 
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==mm/dd/yyyy==
==04/07/2014==
* Insert content here...
Trying golden gate for receiver intermediate. <br>
***Changes*** <br>
Not doing gel extraction and instead using DpnI digest and low concentration of plasmid in PCR.
<br>
PCR
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''No.'''
| align="center" style="background:#f0f0f0;"|'''Template'''
| align="center" style="background:#f0f0f0;"|'''Primer 1'''
| align="center" style="background:#f0f0f0;"|'''Primer 2'''
| align="center" style="background:#f0f0f0;"|'''Annealing Temp'''
|-
| 1||I13522-2 1:100||P111||P112||62
|-
| 2||I13522-3 1:1000||P111||P112||62
|-
| 3||G001||P105||P107||61
|-
| 4||I13522-2 1:100||P103||P104||49
|-
| 5||I13522-3 1:1000||P103||P104||49
|}
<br>
Ran reactions 1,2,3 on Haynes lab PCR machine with annealing temp 61, 3.5 min extension time and 35 cycles.<br>
Ran reactions 4 and 5 on Wang lab PCR machine with annealing temp 49, 1 min ext time and 35 cycles. <br><br>
DpnI digest<br>
Positive control was a miniprepped plasmid at 100ng/ul concentration. Incubated at 37°C for 40 minutes.
{| {{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
|}
 
Only digested reactions 1, 2, 4, and 5. Rxn 3 does not have plasmid DNA, the template is a g-block fragment.
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Thing'''
| align="center" style="background:#f0f0f0;"|'''Volume (ul)'''
|-
| Digest buffer||2
|-
| PCR rxn||17
|-
| DpnI||1
|}
 
<br>
Ran gel of 10 ul of positive DpnI control (should only be cut up stuff, maybe a smear, maybe nothing), 10ul of negative DpnI control (should see uncut plasmid only), 5ul of PCR rxns 1-5 (not DpnI digested)




Revision as of 14:28, 7 April 2014

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04/07/2014

Trying golden gate for receiver intermediate.

      • Changes***

Not doing gel extraction and instead using DpnI digest and low concentration of plasmid in PCR.
PCR

No. Template Primer 1 Primer 2 Annealing Temp
1 I13522-2 1:100 P111 P112 62
2 I13522-3 1:1000 P111 P112 62
3 G001 P105 P107 61
4 I13522-2 1:100 P103 P104 49
5 I13522-3 1:1000 P103 P104 49


Ran reactions 1,2,3 on Haynes lab PCR machine with annealing temp 61, 3.5 min extension time and 35 cycles.
Ran reactions 4 and 5 on Wang lab PCR machine with annealing temp 49, 1 min ext time and 35 cycles.

DpnI digest
Positive control was a miniprepped plasmid at 100ng/ul concentration. Incubated at 37°C for 40 minutes.

' Volume (ul) '
Thing Positive ctrl Negative ctrl
H2O 12 12
Green digest buffer 2 2
pTet mCh 5 5
DpnI 1 0

Only digested reactions 1, 2, 4, and 5. Rxn 3 does not have plasmid DNA, the template is a g-block fragment.

Thing Volume (ul)
Digest buffer 2
PCR rxn 17
DpnI 1


Ran gel of 10 ul of positive DpnI control (should only be cut up stuff, maybe a smear, maybe nothing), 10ul of negative DpnI control (should see uncut plasmid only), 5ul of PCR rxns 1-5 (not DpnI digested)



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