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

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Summary)
(Summary)
(11 intermediate revisions not shown.)
Line 17: Line 17:
*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.
-
*Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
+
* 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.
-
*Pour gel into tray. Wait twenty minutes for gel to settle.
+
*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.
-
*Turn on electrophoresis and let run for thirty minutes.
+
*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>
----
----

Revision as of 03:37, 14 December 2012

Engineering PC-TFs Main project page
Previous entry      Next entry

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



Personal tools