Haynes Lab:Notebook/Engineering PC-TFs/2012/12/10: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Engineering PC-TFs</span>
|style="background-color: #FFCC00"|<br>[[Image:Hayneslab3.gif|200px|center]][[Image:Asu logo 3.gif|200px|center]]<span style="font-size:22px;"><br></span>
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==Summary==
==Summary==
*  
 
* Gel Electrophoresis (PCR Primer Reactions)
 
Follow steps for gel electrophoresis.
*Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
*Create 1% gel by putting .6 grams of agarose into flask.
*Microwave agarose solution for 30 seconds
*Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 30 more seconds.
*When flask is taken out of microwave, make sure that the agarose is completed dissolved.
*Set up wells on gel electrophoresis apparatus. The mold for the first digest is B-10. Use the thin "teeth" and slip in the notches.
* Waited 10-15 minutes for gel to cool. Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
*The gel was poured into tray. Wait twenty minutes for gel to settle.
*Wash the agarose gel flask.
*Pipette 15 μL of DNA ladder into first well. Pipette 5μL of PCR reactions into subsequent wells.
*Turn on electrophoresis (105 V) and let run for thirty minutes.
 
[[Image:December_12_2012.jpg|400px||left|]]
<br><br><br><br><br><br><br><br><br><br>
 
----
*Zymo DNA Clean & Concentrator Kit
 
Follow these steps for Zymo DNA Clean % Concentrator Kit
 
*Add 30 μL of DNA Binding Buffer to each 15 μL PCR Reaction (#1,2,3,4,5)
*Pipette up and down mixture several times to make sure it's thoroughly mixed.
*Centrifuged at top speed for 30 seconds. Flow through was discarded.
*Added 200 μL of Wash Buffer the column and centrifuged for 30 seconds. Repeated step.
*Spin Column for each PCR rxn placed in new 1.5 mL tube.
*30μL of water was direct added to the column to elute DNA.
 
[[Image:December_12_2012plateread.jpg|150px||left|]]
<br><br><br><br><br><br><br><br><br><br><br>
 
----
 
'''PCR of Gibson BioBricks'''
*'''[[User:Karmella Haynes|---Karmella]] 13:55, 10 December 2012 (EST)''': Do not gel purify PCR products. Instead, Zymo DNA Clean & Concentrator kit to clean up the PCR reactions. Elute with 30 μL H<sub>2</sub>O. Measure concentration on the plate reader.
*'''[[User:Karmella Haynes|---Karmella]] 13:55, 10 December 2012 (EST)''': Give me your pSB1A3 so that I can do a retransformation
 




Revision as of 18:32, 6 September 2013



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<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Summary

  • Gel Electrophoresis (PCR Primer Reactions)

Follow steps for gel electrophoresis.

  • Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
  • Create 1% gel by putting .6 grams of agarose into flask.
  • Microwave agarose solution for 30 seconds
  • Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 30 more seconds.
  • When flask is taken out of microwave, make sure that the agarose is completed dissolved.
  • Set up wells on gel electrophoresis apparatus. The mold for the first digest is B-10. Use the thin "teeth" and slip in the notches.
  • Waited 10-15 minutes for gel to cool. Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
  • The gel was poured into tray. Wait twenty minutes for gel to settle.
  • Wash the agarose gel flask.
  • Pipette 15 μL of DNA ladder into first well. Pipette 5μL of PCR reactions into subsequent wells.
  • Turn on electrophoresis (105 V) and let run for thirty minutes.











  • Zymo DNA Clean & Concentrator Kit

Follow these steps for Zymo DNA Clean % Concentrator Kit

  • Add 30 μL of DNA Binding Buffer to each 15 μL PCR Reaction (#1,2,3,4,5)
  • Pipette up and down mixture several times to make sure it's thoroughly mixed.
  • Centrifuged at top speed for 30 seconds. Flow through was discarded.
  • Added 200 μL of Wash Buffer the column and centrifuged for 30 seconds. Repeated step.
  • Spin Column for each PCR rxn placed in new 1.5 mL tube.
  • 30μL of water was direct added to the column to elute DNA.












PCR of Gibson BioBricks

  • ---Karmella 13:55, 10 December 2012 (EST): Do not gel purify PCR products. Instead, Zymo DNA Clean & Concentrator kit to clean up the PCR reactions. Elute with 30 μL H2O. Measure concentration on the plate reader.
  • ---Karmella 13:55, 10 December 2012 (EST): Give me your pSB1A3 so that I can do a retransformation



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