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

From OpenWetWare

< Haynes Lab:Notebook | Jan | 2013 | 09(Difference between revisions)
Jump to: navigation, search
(Plasmid Extraction and Validation of fshPCD)
Current revision (16:13, 11 October 2013) (view source)
(Plasmid Extraction and Validation of fshPCD)
 
Line 12: Line 12:
** Results for Streaks 1 and 2: Growth in both areas along zig-zag line. Bottom left of agar plate dried out. 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 1: fshPCD/V0120 colony 1; BBa_J176002; part = 186 bp; vector = 3200 bp
* Sample 2: fshPCD/V0120 colony 2; same
* Sample 2: fshPCD/V0120 colony 2; same
-
* Sample 3: fshPCD/V0120 (control DNA); BBa_J176002; part = 186 bp; vector = 4986 bp
+
* Sample 3: fshPCD/V0120 (control DNA); BBa_J176002; part = 186 bp; vector = 3200 bp
'''DNA Concentration Data'''
'''DNA Concentration Data'''
Line 49: Line 49:
| bgcolor=#cfcfcf | Reagent  
| bgcolor=#cfcfcf | Reagent  
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Volume
-
| rowspan="7" | <u>Expected:</u><br>1. Plasmid 1 = 186 bp, 4986 <br>2. Plasmid 2 = 186, 4986 <br>
+
| rowspan="7" | <u>Expected:</u><br>1. Plasmid 1 = 186 bp, 3200 <br>2. Plasmid 2 = 186, 3200 <br>
| 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]
| 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]
|-
|-
Line 65: Line 65:
|}
|}
-
'''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.
+
'''Conclusion:''' All samples had a band just under the 5000 bp band (the brightest top band), which was probably the 3200 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. After talking with the professor, a possible explanation is that this band was supercoiled undigested plasmid DNA. It was decided to submit samples 1 and the control DNA for DNA sequencing.
'''DNA Sequencing Samples'''
'''DNA Sequencing Samples'''
<!-- * signs create an automatically bulleted list. Replace 'date' with the date you submitted the samples -->
<!-- * signs create an automatically bulleted list. Replace 'date' with the date you submitted the samples -->
<!-- The Order Number is shown at the top of your sequencing order form. Record in the indicated spot below -->
<!-- The Order Number is shown at the top of your sequencing order form. Record in the indicated spot below -->
-
* Submitted to DNASU on: 'date'
+
* Submitted to DNASU on: '10/8/13'
* Order Number: 8215
* Order Number: 8215
<!-- # 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. -->
<!-- # 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. -->
-
# Plasmid 1 - forward primer 'name'
+
# fshPCD - forward primer 'P0001' 186/186 matches. 100% alignment across the sequence.
-
# Plasmid 1 - reverse primer 'name'
+
# fshPCD - reverse primer 'P0002' 186/186 matches. 100% alignment across the sequence.
-
# Plasmid 2 - forward primer 'name'
+
# fshPCD-control DNA - forward primer 'P0001' 186/186 matches. 100% alignment across the sequence.
-
# Plasmid 2 - reverse primer 'name'
+
# fshPCD-control DNA - reverse primer 'P0002' 186/186 matches. 100% alignment across the sequence.

Current revision

Project name Main project page
Previous entry      Next entry

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 = 3200 bp
  • Sample 2: fshPCD/V0120 colony 2; same
  • Sample 3: fshPCD/V0120 (control DNA); BBa_J176002; part = 186 bp; vector = 3200 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, 3200
2. Plasmid 2 = 186, 3200
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 3200 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. After talking with the professor, a possible explanation is that this band was supercoiled undigested plasmid DNA. It was decided to submit samples 1 and the control DNA for DNA sequencing.

DNA Sequencing Samples

  • Submitted to DNASU on: '10/8/13'
  • Order Number: 8215
  1. fshPCD - forward primer 'P0001' 186/186 matches. 100% alignment across the sequence.
  2. fshPCD - reverse primer 'P0002' 186/186 matches. 100% alignment across the sequence.
  3. fshPCD-control DNA - forward primer 'P0001' 186/186 matches. 100% alignment across the sequence.
  4. fshPCD-control DNA - reverse primer 'P0002' 186/186 matches. 100% alignment across the sequence.




Personal tools