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;"> | |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"| | |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]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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== | ==Overview== | ||
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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. | ||
Followed the instructions in the Sigma Plasmid Miniprep kit. | |||
Sample ID Conc. (ng/ | ==DNA Concentration== | ||
DBN001_pSB1A3 colony 1 78. | |||
DBN001_pSB1A3 colony 2 59. | {| {{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 | |||
|- | |||
| DBN001_pSB1A3 colony 2||59.95||4.37||2.14 | |||
|} | |||
==Restriction Digest== | |||
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| EcoRI||1 | | EcoRI||1 | ||
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| | | SpeI||1 | ||
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| Water||17.3 - 15 | | Water||17.3 - 15 | ||
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Digest for | 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 | ||||||||||||||||||||||||||
OverviewDue to low yield of plasmid from last attempt, am trying again from streaked plates made earlier. NotesOnce 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
Restriction Digest
Digest for 10 minutes at 37°C. Gel ElectrophoresisLost 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. |