Haynes Lab:Notebook/Jan/2013/10/18

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(Entry title)
Current revision (14:21, 9 November 2013) (view source)
(Extraction and Verification of mCh)
 
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| Plasmid || OD260 || OD260/280 || ng/μL
| Plasmid || OD260 || OD260/280 || ng/μL
|-
|-
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| 1. Plasmid 1 || --- || --- || ---
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| 1. Plasmid 1 || 0.12 || 1.84 || 120
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|-
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| 2. Plasmid 2 || --- || --- || ---
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| 2. Plasmid 2 || 0.026 || 1.656 || 26
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| bgcolor=#cfcfcf | Reagent  
| bgcolor=#cfcfcf | Reagent  
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Volume
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| rowspan="7" | <u>Expected:</u><br>1. Plasmid 1 = insert size, vector size<br>2. Plasmid 2 = insert size, vector size<br>
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| rowspan="7" | <u>Expected:</u><br>1. Plasmid 1 = 705 bp, 3200 bp<br>2. Plasmid 2 = 705 bp, 3200 bp<br>
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| rowspan="7" | [[Image:GelImage.jpg|400px|Today's gel]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
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| 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]
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|-
| DNA(plasmid) || 3.0 μL
| DNA(plasmid) || 3.0 μL
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| &nbsp; || 15 μL --> 37°C/ 15 min.
| &nbsp; || 15 μL --> 37°C/ 15 min.
|}
|}
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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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<!-- The Order Number is shown at the top of your sequencing order form. Record in the indicated spot below -->
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* Submitted to DNASU on: 'date'
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* Submitted to DNASU on: '10/21/13'
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* Order Number:  
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* 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. -->
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# Plasmid 1 - forward primer 'name'
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# mCh - forward primer 'P0001' -- 705 matches, thus 100% matching across the sequence. DNA cloning successful.
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# Plasmid 1 - reverse primer 'name'
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# mCh - reverse primer 'P0002' -- 705 matches, so 100% match. DNA cloning successful.
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# Plasmid 2 - forward primer 'name'
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# Plasmid 2 - reverse primer 'name'
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Current revision

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Extraction and Verification of mCh

  • Name: mCh / BBa_J176005
    • Date of culture inoculation: 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

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

  • Checked plasmid minipreps with EcoRI/PstI digests
Reagent Volume Expected:
1. Plasmid 1 = 705 bp, 3200 bp
2. Plasmid 2 = 705 bp, 3200 bp
Today's gel
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 3.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 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

  • Submitted to DNASU on: '10/21/13'
  • Order Number: 8258
  1. mCh - forward primer 'P0001' -- 705 matches, thus 100% matching across the sequence. DNA cloning successful.
  2. mCh - reverse primer 'P0002' -- 705 matches, so 100% match. DNA cloning successful.



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