Haynes Lab:Notebook/Jan/2013/09/27

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(Plasmid Extraction and Validation of fshPCD)
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==Entry title==
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==Plasmid Extraction and Validation of fshPCD==
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* Name: fshPCD / BBa_J176002
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** Date of culture inoculation: [http://openwetware.org/wiki/Haynes_Lab:Notebook/Jan/2013/09/26 9/26/13]
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** Results for Cultures 1 and 2: Mixture is cloudy so growth in both. Success.
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** Results for Streaks 1 and 2: Growth in both areas along zig-zag line. Bottom left of agar plate dried out. Success.
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* Sample 1: fshPCD/V0120 colony 1; BBa_J176002; part = 186 bp; vector = 4986 bp
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* Sample 2: fshPCD/V0120 colony 2; same
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* Sample 3: fshPCD/V0120 (control DNA); BBa_J176002; part = 186 bp; vector = 4986 bp
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'''DNA Concentration Data'''
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{| {{table}}
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| Plasmid || OD260 || OD260/280 || ng/μL
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|-
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| 1. Plasmid 1 || 0.186 || 1.859 || 185.784
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|-
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| 2. Plasmid 2 || 0.138 || 1.827 || 138.008
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|}
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'''Restriction Digest Table'''<br>
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* Checked plasmid minipreps with EcoRI/PstI digests
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{| {{table}} border="1" cellspacing="3"
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<!-- The next two "rowspan=7" cells span  all 7 rows in the table so that they can fit the "Expected" list and a gel image. rowspan is how you create merged rows in Wiki code. -->
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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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|- valign="top"
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| bgcolor=#cfcfcf | Reagent
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| bgcolor=#cfcfcf | Volume
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| rowspan="7" | <u>Expected:</u><br>1. Plasmid 1 = 186 bp, 4986 <br>2. Plasmid 2 = 186, 4986 <br>
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| rowspan="7" | [[Image:Jan Simper fshPCD.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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|-
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| DNA(plasmid) || 3.0 μL
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|-
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| 10X buffer || 1.5
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|-
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| EcoRI || 1.0
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|-
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| PstI || 1.0
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|-
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| dH<sub>2</sub>O || 8.5
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|-
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| &nbsp; || 15 μL --> 37°C/ 15 min.
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|}
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'''Conclusion:''' All samples had a band just under the 5000 bp band (the brightest top band), which was probably the 4986 bp vector. Also, the expected part base pair was 186 bp, and the band in the picture is slightly above the 200 bp band, but all three samples including the control DNA had the band at the same location, so overall, the restriction digest was successful. However, additional help about the two bands around the 1500 bp location that appeared in samples 1 and 2 but not in the control DNA will be received before continuing.
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'''DNA Sequencing Samples'''
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* Submitted to DNASU on: 'date'
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* Order Number:
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<!-- # 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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# Plasmid 1 - reverse primer 'name'
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# Plasmid 2 - forward primer 'name'
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# Plasmid 2 - reverse primer 'name'
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Revision as of 19:26, 4 October 2013

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Plasmid Extraction and Validation of fshPCD

  • Name: fshPCD / BBa_J176002
    • Date of culture inoculation: 9/26/13
    • 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. Bottom left of agar plate dried out. Success.
  • Sample 1: fshPCD/V0120 colony 1; BBa_J176002; part = 186 bp; vector = 4986 bp
  • Sample 2: fshPCD/V0120 colony 2; same
  • Sample 3: fshPCD/V0120 (control DNA); BBa_J176002; part = 186 bp; vector = 4986 bp

DNA Concentration Data

Plasmid OD260 OD260/280 ng/μL
1. Plasmid 1 0.186 1.859 185.784
2. Plasmid 2 0.138 1.827 138.008


Restriction Digest Table

  • Checked plasmid minipreps with EcoRI/PstI digests
Reagent Volume Expected:
1. Plasmid 1 = 186 bp, 4986
2. Plasmid 2 = 186, 4986
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.

Conclusion: All samples had a band just under the 5000 bp band (the brightest top band), which was probably the 4986 bp vector. Also, the expected part base pair was 186 bp, and the band in the picture is slightly above the 200 bp band, but all three samples including the control DNA had the band at the same location, so overall, the restriction digest was successful. However, additional help about the two bands around the 1500 bp location that appeared in samples 1 and 2 but not in the control DNA will be received before continuing.

DNA Sequencing Samples

  • Submitted to DNASU on: 'date'
  • Order Number:
  1. Plasmid 1 - forward primer 'name'
  2. Plasmid 1 - reverse primer 'name'
  3. Plasmid 2 - forward primer 'name'
  4. Plasmid 2 - reverse primer 'name'




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