Haynes Lab:Notebook/Engineering PC-TFs/2012/12/10: Difference between revisions
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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> | |||
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*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|]] | |||
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Revision as of 01:37, 14 December 2012
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Summary
Follow steps for gel electrophoresis.
Follow these steps for Zymo DNA Clean % Concentrator Kit
PCR of Gibson BioBricks
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