Haynes Lab:Notebook/Jan/2013/10/18: 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;"> Project name</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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== | ==Extraction and Verification of mCh== | ||
* | * Name: mCh / BBa_J176005 | ||
** Date of culture inoculation: [http://openwetware.org/wiki/Haynes_Lab:Notebook/Jan/2013/09/26 10/17/13] | |||
** Results for Cultures 1 and 2: Mixture is cloudy so growth in both. Success. | |||
** Results for Streaks 1 and 2: Growth in both areas along zig-zag line, although more growth in colony 1 streak than colony 2 streak. Overall success. | |||
* Sample 1: mCh/V0120 colony 1; BBa_J176005; part = 705 bp; vector = 3200 bp | |||
* Sample 2: mCh/V0120 colony 2; same | |||
* Sample 3: mCh/V0120 (control DNA); BBa_J176005; part = 705 bp; vector = 3200 bp | |||
'''DNA Concentration Data''' | |||
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{| {{table}} | |||
|- bgcolor=#cfcfcf | |||
| Plasmid || OD260 || OD260/280 || ng/μL | |||
|- | |||
| 1. Plasmid 1 || 0.12 || 1.84 || 120 | |||
|- | |||
| 2. Plasmid 2 || 0.026 || 1.656 || 26 | |||
|} | |||
'''Restriction Digest Table'''<br> | |||
* Checked plasmid minipreps with EcoRI/PstI digests | |||
{| {{table}} border="1" cellspacing="3" | |||
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<!-- Replace Plasmid 1 and 2 with the names of your plasmids. Replace insert size and vector size with appropriate information for your plasmids. If you only have one Plasmid, delete all the text for Plasmid 2 | |||
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| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Volume | |||
| rowspan="7" | <u>Expected:</u><br>1. Plasmid 1 = 705 bp, 3200 bp<br>2. Plasmid 2 = 705 bp, 3200 bp<br> | |||
| rowspan="7" | [[Image:Jan_Simper_mCh_DNA_Boot_Camp.jpg|400px|Today's gel]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|- | |||
| DNA(plasmid) || 3.0 μL | |||
|- | |||
| 10X buffer || 1.5 | |||
|- | |||
| EcoRI || 1.0 | |||
|- | |||
| PstI || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || 8.5 | |||
|- | |||
| || 15 μL --> 37°C/ 15 min. | |||
|} | |||
Both samples 1 and 3 had a band slightly above the 3000 bp band, which was probably the 3200 bp vector. In addition, both samples 1 and 3 had a band slightly above the 700 bp band, which was likely the 705 bp plasmid. Overall, there is evidence to conclude that Sample 1 is the cloned plasmid DNA because of its similarities to the control DNA. Sample 2 had no bands of any sort and there was likely no DNA there in the first place due to a bad miniprep. Sample 1 was submitted for DNA sequencing to confirm that it is in fact the cloned plasmid DNA. | |||
'''DNA Sequencing Samples''' | |||
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* Submitted to DNASU on: '10/21/13' | |||
* Order Number: 8258 | |||
<!-- # signs create an automatically numbered list. Replace Plasmid 1 & 2 with the names of your plasmids. Replace 'name' with the appropriate name of each primer. --> | |||
# mCh - forward primer 'P0001' -- 705 matches, thus 100% matching across the sequence. DNA cloning successful. | |||
# mCh - reverse primer 'P0002' -- 705 matches, so 100% match. DNA cloning successful. | |||
Latest revision as of 23:29, 26 September 2017
Project name | Main project page Previous entry Next entry | ||||||||||||||||||||||||||||
Extraction and Verification of mCh
Both samples 1 and 3 had a band slightly above the 3000 bp band, which was probably the 3200 bp vector. In addition, both samples 1 and 3 had a band slightly above the 700 bp band, which was likely the 705 bp plasmid. Overall, there is evidence to conclude that Sample 1 is the cloned plasmid DNA because of its similarities to the control DNA. Sample 2 had no bands of any sort and there was likely no DNA there in the first place due to a bad miniprep. Sample 1 was submitted for DNA sequencing to confirm that it is in fact the cloned plasmid DNA.
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