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

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(Autocreate 2014/05/14 Entry for Haynes_Lab:Notebook/Characterizing_AHL_quorum_sensing_homologs)
 
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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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''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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Set up PCR for EsaR and EsaI
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Template'''
| align="center" style="background:#f0f0f0;"|'''F Primer'''
| align="center" style="background:#f0f0f0;"|'''R Primer'''
| align="center" style="background:#f0f0f0;"|'''Annealing T'''
| align="center" style="background:#f0f0f0;"|'''Expected length'''
|-
| EsaR kan||P109||P110||38||
|-
| EsaI Sender||P128||P129||54||
|-
| EsaI Sender||P128||P129||54||
|}


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Revision as of 10:49, 14 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.

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


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