Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2014/05/14

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Current revision (11:05, 15 May 2014) (view source)
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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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{| {{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
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|}
<br><br>
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''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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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 vec0.0570.031.90356.995
LasI0.030.0142.05829.695
RhlI0.0260.0131.99226.111



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 kanP109P11038
EsaI SenderP128P12954
EsaI SenderP128P12954



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