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

From OpenWetWare
Jump to navigationJump to search
Line 8: Line 8:
==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.  
<br>
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sample Read#'''
| align="center" style="background:#f0f0f0;"|'''260'''
| align="center" style="background:#f0f0f0;"|'''280'''
| align="center" style="background:#f0f0f0;"|'''260/280'''
| align="center" style="background:#f0f0f0;"|'''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
|}
<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''
Line 25: Line 39:
| EsaI Sender||P128||P129||54||
| EsaI Sender||P128||P129||54||
|}
|}


<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Revision as of 09:05, 15 May 2014

Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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



Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/Characterizing_AHL_quorum_sensing_homologs/2014/05/14&oldid=793417"