Haynes Lab:Notebook/Engineering PC-TFs/2012/12/10: Difference between revisions
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|style="background-color: # | |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''' | '''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 | |||
Latest revision as of 22:19, 26 September 2017
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Summary
Follow steps for gel electrophoresis.
Follow these steps for Zymo DNA Clean % Concentrator Kit
PCR of Gibson BioBricks
|