Team:EPF-Lausanne/Notebook/3 August 2012

From 2012.igem.org

(Difference between revisions)
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PMP2 control for plasmid
PMP2 control for plasmid
-
DNA 2µL
+
*DNA 2µL
-
HindIII 1µL
+
*HindIII 1µL
-
XbaI 1µL
+
*XbaI 1µL
-
N4 buffer 10x 5µL
+
*N4 buffer 10x 5µL
-
BSA 100x 0.5µL
+
*BSA 100x 0.5µL
-
Water 40.5 µL
+
*Water 40.5 µL
PMP2 control for NotI
PMP2 control for NotI
-
DNA 2µL
+
*DNA 2µL
-
NotI 1µL
+
*NotI 1µL
-
N4 buffer 10x 5µL
+
*N4 buffer 10x 5µL
-
BSA 100x 0.5µL
+
*BSA 100x 0.5µL
-
Water 41.5 µL
+
*Water 41.5 µL
PMP2 control for SpeI
PMP2 control for SpeI
-
DNA 2µL
+
*DNA 2µL
-
SpeI 1µL
+
*SpeI 1µL
-
N4 buffer 10x 5µL
+
*N4 buffer 10x 5µL
-
BSA 100x 0.5µL
+
*BSA 100x 0.5µL
-
Water 41.5 µL
+
*Water 41.5 µL
PMP2 for ligation
PMP2 for ligation
-
DNA 19µL
+
*DNA 19µL
-
SpeI 1µL
+
*SpeI 1µL
-
NotI 1µL
+
*NotI 1µL
-
N2 buffer 10x 5µL
+
*N2 buffer 10x 5µL
-
BSA 100x 0.5µL
+
*BSA 100x 0.5µL
-
Water 23.5 µL
+
*Water 23.5 µL
PGL2 for ligation
PGL2 for ligation
-
DNA 12µL
+
*DNA 12µL
-
HindIII 1µL
+
*HindIII 1µL
-
MfeI 1µL
+
*MfeI 1µL
-
N2 buffer 10x 5µL
+
*N2 buffer 10x 5µL
-
BSA 100x 0.5µL
+
*BSA 100x 0.5µL
-
Water 31 µL
+
*Water 31 µL
PGL3 for ligation
PGL3 for ligation
-
DNA 16µL
+
*DNA 16µL
-
HindIII 1µL
+
*HindIII 1µL
-
FseI 1µL
+
*FseI 1µL
-
N2 buffer 10x 5µL
+
*N2 buffer 10x 5µL
-
BSA 100x 0.5µL
+
*BSA 100x 0.5µL
-
Water 26.5 µL
+
*Water 26.5 µL
All let 1:30 hours at 37ºC, shaking (600 rpm).
All let 1:30 hours at 37ºC, shaking (600 rpm).
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</style></head><body dir="ltr" style="writing-mode:lr-tb; "><table border="0" cellspacing="0" cellpadding="0" class="ta1"><colgroup><col width="99"/><col width="283"/><col width="200"/><col width="216"/><col width="193"/><col width="164"/><col width="212"/><col width="159"/><col width="221"/><col width="231"/></colgroup><tr class="ro1"><td style="text-align:left;width:0.889in; " class="Default"><p>ladder </p></td><td style="text-align:left;width:2.548in; " class="Default"> </td><td style="text-align:left;width:1.802in; " class="Default"><p>PMP2 cut w/ NotI SpeI</p></td><td style="text-align:left;width:1.9425in; " class="Default"> </td><td style="text-align:left;width:1.7402in; " class="Default"><p>PGL2 cut w/ HindII MfeI</p></td><td style="text-align:left;width:1.4756in; " class="Default"> </td><td style="text-align:left;width:1.911in; " class="Default"><p>PGL3 cut w/ HindII FseI</p></td><td style="text-align:left;width:1.4291in; " class="Default"> </td><td style="text-align:left;width:1.989in; " class="Default"><p>control of HindII w/ PGL3</p></td><td style="text-align:left;width:2.0819in; " class="Default"><p>control of FseI w/ PGL3</p></td></tr><tr class="ro2"><td style="text-align:left;width:0.889in; " class="Default"> </td><td style="text-align:left;width:2.548in; " class="Default"> </td><td style="text-align:left;width:1.802in; " class="Default"> </td><td style="text-align:left;width:1.9425in; " class="Default"> </td><td style="text-align:left;width:1.7402in; " class="Default"> </td><td style="text-align:left;width:1.4756in; " class="Default"> </td><td style="text-align:left;width:1.911in; " class="Default"> </td><td style="text-align:left;width:1.4291in; " class="Default"> </td><td style="text-align:left;width:1.989in; " class="Default"> </td><td style="text-align:left;width:2.0819in; " class="Default"> </td></tr><tr class="ro1"><td style="text-align:left;width:0.889in; " class="Default"><p>ladder </p></td><td style="text-align:left;width:2.548in; " class="Default"><p>control w/ HindIII and XbaI for PMP2</p></td><td style="text-align:left;width:1.802in; " class="Default"><p>Control of NotI w/ PMP2</p></td><td style="text-align:left;width:1.9425in; " class="Default"><p>Control of SpeI w/ PMP2</p></td><td style="text-align:left;width:1.7402in; " class="Default"> </td><td style="text-align:left;width:1.4756in; " class="Default"><p>eGFP pcr product</p></td><td style="text-align:left;width:1.911in; " class="Default"><p>TNFR pcr product</p></td><td style="text-align:left;width:1.4291in; " class="Default"><p>SEAP pcr product</p></td><td style="text-align:left;width:1.989in; " class="Default"> </td><td style="text-align:left;width:2.0819in; " class="Default"> </td></tr></table><table border="0" cellspacing="0" cellpadding="0" class="ta1"><colgroup><col width="99"/></colgroup><tr class="ro2"><td style="text-align:left;width:0.889in; " class="Default"> </td></tr></table><table border="0" cellspacing="0" cellpadding="0" class="ta1"><colgroup><col width="99"/></colgroup><tr class="ro2"><td style="text-align:left;width:0.889in; " class="Default"> </td></tr></table></body></html>
</style></head><body dir="ltr" style="writing-mode:lr-tb; "><table border="0" cellspacing="0" cellpadding="0" class="ta1"><colgroup><col width="99"/><col width="283"/><col width="200"/><col width="216"/><col width="193"/><col width="164"/><col width="212"/><col width="159"/><col width="221"/><col width="231"/></colgroup><tr class="ro1"><td style="text-align:left;width:0.889in; " class="Default"><p>ladder </p></td><td style="text-align:left;width:2.548in; " class="Default"> </td><td style="text-align:left;width:1.802in; " class="Default"><p>PMP2 cut w/ NotI SpeI</p></td><td style="text-align:left;width:1.9425in; " class="Default"> </td><td style="text-align:left;width:1.7402in; " class="Default"><p>PGL2 cut w/ HindII MfeI</p></td><td style="text-align:left;width:1.4756in; " class="Default"> </td><td style="text-align:left;width:1.911in; " class="Default"><p>PGL3 cut w/ HindII FseI</p></td><td style="text-align:left;width:1.4291in; " class="Default"> </td><td style="text-align:left;width:1.989in; " class="Default"><p>control of HindII w/ PGL3</p></td><td style="text-align:left;width:2.0819in; " class="Default"><p>control of FseI w/ PGL3</p></td></tr><tr class="ro2"><td style="text-align:left;width:0.889in; " class="Default"> </td><td style="text-align:left;width:2.548in; " class="Default"> </td><td style="text-align:left;width:1.802in; " class="Default"> </td><td style="text-align:left;width:1.9425in; " class="Default"> </td><td style="text-align:left;width:1.7402in; " class="Default"> </td><td style="text-align:left;width:1.4756in; " class="Default"> </td><td style="text-align:left;width:1.911in; " class="Default"> </td><td style="text-align:left;width:1.4291in; " class="Default"> </td><td style="text-align:left;width:1.989in; " class="Default"> </td><td style="text-align:left;width:2.0819in; " class="Default"> </td></tr><tr class="ro1"><td style="text-align:left;width:0.889in; " class="Default"><p>ladder </p></td><td style="text-align:left;width:2.548in; " class="Default"><p>control w/ HindIII and XbaI for PMP2</p></td><td style="text-align:left;width:1.802in; " class="Default"><p>Control of NotI w/ PMP2</p></td><td style="text-align:left;width:1.9425in; " class="Default"><p>Control of SpeI w/ PMP2</p></td><td style="text-align:left;width:1.7402in; " class="Default"> </td><td style="text-align:left;width:1.4756in; " class="Default"><p>eGFP pcr product</p></td><td style="text-align:left;width:1.911in; " class="Default"><p>TNFR pcr product</p></td><td style="text-align:left;width:1.4291in; " class="Default"><p>SEAP pcr product</p></td><td style="text-align:left;width:1.989in; " class="Default"> </td><td style="text-align:left;width:2.0819in; " class="Default"> </td></tr></table><table border="0" cellspacing="0" cellpadding="0" class="ta1"><colgroup><col width="99"/></colgroup><tr class="ro2"><td style="text-align:left;width:0.889in; " class="Default"> </td></tr></table><table border="0" cellspacing="0" cellpadding="0" class="ta1"><colgroup><col width="99"/></colgroup><tr class="ro2"><td style="text-align:left;width:0.889in; " class="Default"> </td></tr></table></body></html>
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Revision as of 14:10, 6 August 2012



Digestion of PMP and PGL4.30 for ligation

prepared PMP2 , PGL2 and PGL3 for ligation

restriction enzyme digest mixtures were as follows:

PMP2 control for plasmid

  • DNA 2µL
  • HindIII 1µL
  • XbaI 1µL
  • N4 buffer 10x 5µL
  • BSA 100x 0.5µL
  • Water 40.5 µL

PMP2 control for NotI

  • DNA 2µL
  • NotI 1µL
  • N4 buffer 10x 5µL
  • BSA 100x 0.5µL
  • Water 41.5 µL

PMP2 control for SpeI

  • DNA 2µL
  • SpeI 1µL
  • N4 buffer 10x 5µL
  • BSA 100x 0.5µL
  • Water 41.5 µL


PMP2 for ligation

  • DNA 19µL
  • SpeI 1µL
  • NotI 1µL
  • N2 buffer 10x 5µL
  • BSA 100x 0.5µL
  • Water 23.5 µL

PGL2 for ligation

  • DNA 12µL
  • HindIII 1µL
  • MfeI 1µL
  • N2 buffer 10x 5µL
  • BSA 100x 0.5µL
  • Water 31 µL

PGL3 for ligation

  • DNA 16µL
  • HindIII 1µL
  • FseI 1µL
  • N2 buffer 10x 5µL
  • BSA 100x 0.5µL
  • Water 26.5 µL

All let 1:30 hours at 37ºC, shaking (600 rpm).


Bands got in the control of DNA and enzymes. Everything seems to be correct now!

After we ran the digestion products in a gel (1% agarose, 50 µl of digestion product + 10 µl of loading dye).


Gel with digested backbones and pcr products for readouts



<?xml version="1.0" encoding="UTF-8"?> <!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.1 plus MathML 2.0//EN" "http://www.w3.org/Math/DTD/mathml2/xhtml-math11-f.dtd"> - no title specified

ladder

 

PMP2 cut w/ NotI SpeI

 

PGL2 cut w/ HindII MfeI

 

PGL3 cut w/ HindII FseI

 

control of HindII w/ PGL3

control of FseI w/ PGL3

          

ladder

control w/ HindIII and XbaI for PMP2

Control of NotI w/ PMP2

Control of SpeI w/ PMP2

 

eGFP pcr product

TNFR pcr product

SEAP pcr product

  
 
 




Protocol: None

Forgot to insert protocol.


Comments

Insert comments about what happened.


Wiki Links

Important pages

Pages

Retrieved from "http://2012.igem.org/Team:EPF-Lausanne/Notebook/3_August_2012"