IGEM:Arizona State/2014/Notebook/asu igem 2014/2014/07/14

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==Entry title==
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==TesA, RBS Digests==
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* Made LB agar plates
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# Made LB agar plates
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1 L of distilled water, 35 grams of LB agar powder. Autoclave for 30 minutes.
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#*1 L of distilled water, 35 grams of LB agar powder. Autoclave for 30 minutes.
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# Ran Digestion of TesA and RBS
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#*Cut TesA at X, P and RBS at S, P.
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#*After the digestion, the DNA was extracted, taking the 651 bp  TesA part from both iGEM and Haynes PstI lanes. RBS was also extracted from both sets of lanes (only vector remained).
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#*DNA was stored in the freezer overnight to await ligation
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* Ran Digestion of TesA and RBS
 
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TesA cut at X, P and RBS cut at S, P.
 
Note: in order to ensure the viability of our PstI enzyme, we ran a control with some PstI from Haynes lab. Results are shown below. the PstI of Haynes lab had full digestion, while the iGEM PstI had a small amount of partial digestion, as made manifest by the three bands of the TesA in lane 2 of the gel.
Note: in order to ensure the viability of our PstI enzyme, we ran a control with some PstI from Haynes lab. Results are shown below. the PstI of Haynes lab had full digestion, while the iGEM PstI had a small amount of partial digestion, as made manifest by the three bands of the TesA in lane 2 of the gel.
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| bgcolor=#cfcfcf | TesA (μL)
| bgcolor=#cfcfcf | TesA (μL)
| rowspan="7" | <u>'''Lanes'''</u><br>Our PstI:<br>1. 1kb ladder<br>2. TesA (651 bp)<br> 3. RBS (2083)<br>Haynes PstI:<br> 4. TesA (651 bp)<br> 5. RBS (2083 bp)<br> 6. Repeat of 5 <br>
| rowspan="7" | <u>'''Lanes'''</u><br>Our PstI:<br>1. 1kb ladder<br>2. TesA (651 bp)<br> 3. RBS (2083)<br>Haynes PstI:<br> 4. TesA (651 bp)<br> 5. RBS (2083 bp)<br> 6. Repeat of 5 <br>
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| rowspan="7" | [[image:14_gel_.jpg|400px|gel]]<br> [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
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| rowspan="7" | [[image:14_gel_.jpg|400px|gel]]<br> 15 μL/lane; 1% agarose;[http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
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| DNA(plasmid) || 10.0 || 10.0
| DNA(plasmid) || 10.0 || 10.0

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TesA, RBS Digests

  1. Made LB agar plates
    • 1 L of distilled water, 35 grams of LB agar powder. Autoclave for 30 minutes.
  2. Ran Digestion of TesA and RBS
    • Cut TesA at X, P and RBS at S, P.
    • After the digestion, the DNA was extracted, taking the 651 bp TesA part from both iGEM and Haynes PstI lanes. RBS was also extracted from both sets of lanes (only vector remained).
    • DNA was stored in the freezer overnight to await ligation

Note: in order to ensure the viability of our PstI enzyme, we ran a control with some PstI from Haynes lab. Results are shown below. the PstI of Haynes lab had full digestion, while the iGEM PstI had a small amount of partial digestion, as made manifest by the three bands of the TesA in lane 2 of the gel.


Reagent RBS (μL) TesA (μL) Lanes
Our PstI:
1. 1kb ladder
2. TesA (651 bp)
3. RBS (2083)
Haynes PstI:
4. TesA (651 bp)
5. RBS (2083 bp)
6. Repeat of 5
gel
15 μL/lane; 1% agarose;Ladder
DNA(plasmid) 10.0 10.0
10X FD buffer 2.0 2.0
Enzyme # 1 SpeI: 1.0 XbaI: 1.0
PstI 1.0 1.0
dH2O 6.0 6.0
  20 μL @ 42°C/ ~1 Hr.


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