Difference between revisions of "Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2014/05/14"

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(mm/dd/yyyy)
(mm/dd/yyyy)
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==mm/dd/yyyy==
 
==mm/dd/yyyy==
 
DpnI digest of RhlI PCR from 5/6/14, LasI PCR from 5/12/14, and Receiver Vector Intermediate PCRs 1-3 from 5/13/14. Reaction was 35ul of PCR DNA, 1ul DpnI, and 4ul of Fast Digest Buffer without dye. 47 minutes at 37°C and heat inactivated for 5 minutes at 80°C. Purified with zymo DNA clean and concentration-5 kit (columns from gel extraction kit). Added 150ul of DNA binding buffer to DpnI reactions.  
 
DpnI digest of RhlI PCR from 5/6/14, LasI PCR from 5/12/14, and Receiver Vector Intermediate PCRs 1-3 from 5/13/14. Reaction was 35ul of PCR DNA, 1ul DpnI, and 4ul of Fast Digest Buffer without dye. 47 minutes at 37°C and heat inactivated for 5 minutes at 80°C. Purified with zymo DNA clean and concentration-5 kit (columns from gel extraction kit). Added 150ul of DNA binding buffer to DpnI reactions.  
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<br>
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''Sample Read#'''
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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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| Receiver int vec||0.057||0.03||1.903||56.995
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|-
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| LasI||0.03||0.014||2.058||29.695
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|-
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| RhlI||0.026||0.013||1.992||26.111
 +
|}
 
<br><br>
 
<br><br>
 
''Important change: last time, I gel extracted the Receiver intermediate vector because one of the primers has two binding sites. The subsequence golden gate ligation was unsuccessful so I'm trying it again without gel extracting. I will need to make sure the bands are the correct sizes before sending out for sequencing''
 
''Important change: last time, I gel extracted the Receiver intermediate vector because one of the primers has two binding sites. The subsequence golden gate ligation was unsuccessful so I'm trying it again without gel extracting. I will need to make sure the bands are the correct sizes before sending out for sequencing''
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| EsaI Sender||P128||P129||54||
 
| EsaI Sender||P128||P129||54||
 
|}
 
|}
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Revision as of 08:05, 15 May 2014

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mm/dd/yyyy

DpnI digest of RhlI PCR from 5/6/14, LasI PCR from 5/12/14, and Receiver Vector Intermediate PCRs 1-3 from 5/13/14. Reaction was 35ul of PCR DNA, 1ul DpnI, and 4ul of Fast Digest Buffer without dye. 47 minutes at 37°C and heat inactivated for 5 minutes at 80°C. Purified with zymo DNA clean and concentration-5 kit (columns from gel extraction kit). Added 150ul of DNA binding buffer to DpnI reactions.

Sample Read# 260 280 260/280 ng/µL
Receiver int vec 0.057 0.03 1.903 56.995
LasI 0.03 0.014 2.058 29.695
RhlI 0.026 0.013 1.992 26.111



Important change: last time, I gel extracted the Receiver intermediate vector because one of the primers has two binding sites. The subsequence golden gate ligation was unsuccessful so I'm trying it again without gel extracting. I will need to make sure the bands are the correct sizes before sending out for sequencing

Set up PCR for EsaR and EsaI

Template F Primer R Primer Annealing T Expected length
EsaR kan P109 P110 38
EsaI Sender P128 P129 54
EsaI Sender P128 P129 54