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

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Engineering PC-TFs</span>
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|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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|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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|style="background-color: #800000" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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*When flask is taken out of microwave, make sure that the agarose is completed dissolved.
*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.
*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.
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*Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
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* 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.
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*Pour gel into tray. Wait twenty minutes for gel to settle.
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*The gel was poured into tray. Wait twenty minutes for gel to settle.
*Wash the agarose gel flask.
*Wash the agarose gel flask.
*Pipette 15 μL of DNA ladder into first well. Pipette 5μL of PCR reactions into subsequent wells.
*Pipette 15 μL of DNA ladder into first well. Pipette 5μL of PCR reactions into subsequent wells.
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*Turn on electrophoresis and let run for thirty minutes.
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*Turn on electrophoresis (105 V) and let run for thirty minutes.
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[[Image:December_12_2012.jpg|400px||left|]]
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<br><br><br><br><br><br><br><br><br><br>
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----
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*Zymo DNA Clean & Concentrator Kit
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Follow these steps for Zymo DNA Clean % Concentrator Kit
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*Add 30 μL of DNA Binding Buffer to each 15 μL PCR Reaction (#1,2,3,4,5)
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*Pipette up and down mixture several times to make sure it's thoroughly mixed.
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*Centrifuged at top speed for 30 seconds. Flow through was discarded.
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*Added 200 μL of Wash Buffer the column and centrifuged for 30 seconds. Repeated step.
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*Spin Column for each PCR rxn placed in new 1.5 mL tube.
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*30μL of water was direct added to the column to elute DNA.
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[[Image:December_12_2012plateread.jpg|150px||left|]]
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<br><br><br><br><br><br><br><br><br><br><br>
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Current revision



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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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