Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/17: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Plasmid Miniprep of DBN001_pSB1A3 (2nd attempt)</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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==Plasmid Miniprep of DBN001_pSB1A3 (repeat)==
==Overview==
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<b>--Notes--</b>
Due to low yield of plasmid from last attempt, am trying again from streaked plates made earlier.
 
==Notes==


Once again, liquid culture density is very low. Suspect it's because there's no actual plasmid present.
Once again, liquid culture density is very low. Suspect it's because there's no actual plasmid present.


<b>--DNA Concentration--</b>
Followed the instructions in the Sigma Plasmid Miniprep kit.


Sample ID Conc. (ng/uL) St Dev (ng/uL) 260/280
==DNA Concentration==
DBN001_pSB1A3 colony 1 78.3 4.98 1.89
 
DBN001_pSB1A3 colony 2 59.9 4.37 2.14
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sample ID'''
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/µL)'''
| align="center" style="background:#f0f0f0;"|'''St Dev (ng/µL)'''
| align="center" style="background:#f0f0f0;"|'''260/280'''
|-
| DBN001_pSB1A3 colony 1||78.27||4.98||1.89
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| DBN001_pSB1A3 colony 2||59.95||4.37||2.14
|}


<b>--Restriction Digest--</b>
==Restriction Digest==


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| EcoRI||1
| EcoRI||1
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| PstI||1
| SpeI||1
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| Water||17.3 - 15
| Water||17.3 - 15
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Digest for 15 minutes at 37°C.
Digest for 10 minutes at 37°C.
 
==Gel Electrophoresis==
 
Lost most of sample 1 while pipetting into the lane. Bands will probably be very faint.
 
Expected size fragments from EcoRI/SpeI digestion: 2132, 224
 
[[Image:2015-04-17_DBN001_pSB1A3_digest_annotated.png]]


Still no trace of plasmid present. Going to assume cloning did not work. Will try again next week with fresh phosphatase & ligase, using dephosphorylated backbone as another control.


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Latest revision as of 00:55, 27 September 2017

Plasmid Miniprep of DBN001_pSB1A3 (2nd attempt) Main project page
Previous entry      Next entry

Overview

Due to low yield of plasmid from last attempt, am trying again from streaked plates made earlier.

Notes

Once again, liquid culture density is very low. Suspect it's because there's no actual plasmid present.

Followed the instructions in the Sigma Plasmid Miniprep kit.

DNA Concentration

Sample ID Conc. (ng/µL) St Dev (ng/µL) 260/280
DBN001_pSB1A3 colony 1 78.27 4.98 1.89
DBN001_pSB1A3 colony 2 59.95 4.37 2.14

Restriction Digest

Reagent Volume
DNA 600ng (7.7 - 10 µL)
10x FastDigest Buffer 3
EcoRI 1
SpeI 1
Water 17.3 - 15
Total 30

Digest for 10 minutes at 37°C.

Gel Electrophoresis

Lost most of sample 1 while pipetting into the lane. Bands will probably be very faint.

Expected size fragments from EcoRI/SpeI digestion: 2132, 224

Still no trace of plasmid present. Going to assume cloning did not work. Will try again next week with fresh phosphatase & ligase, using dephosphorylated backbone as another control.


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