Haynes Lab:Notebook/Engineering PC-TFs/2013/09/26: Difference between revisions
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== | ==September 26, 2013== | ||
''Restriction Digest'' | |||
*Perform restriction digest for CMV promoter | *Perform restriction digest for CMV promoter | ||
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| PstI|| 1.0 μL | | PstI|| 1.0 μL | ||
|- | |- | ||
| Green 10x FastDigest buffer || | | Green 10x FastDigest buffer || 0μL | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 18 μL | ||
|- | |- | ||
| || 30 μL --> 37°C/ ~10 min. | | || 30 μL --> 37°C/ ~10 min. | ||
|} | |} | ||
''PEG/MgCl<sub>2</sub> Precipitation'' | |||
* This procedure was adapated from [http://openwetware.org/wiki/Protocol_Size_selective_DNA_precipitation_by_PEG/MgCl2 Size selective DNA precipitation] | |||
*Measure the following into 1.5 mL tubes. | *Measure the following into 1.5 mL tubes. | ||
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| Total || 100 μL || 100 μL|| 100 μL|| 100 μL|| 100 μL | | Total || 100 μL || 100 μL|| 100 μL|| 100 μL|| 100 μL | ||
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*Centrifuge for 15 minutes at 10,000g. | |||
''DNA Clean & Concentrator'' | |||
* Although there was no pellet, ran a DNA Clean & Concentrator on samples. | |||
**Add 2 volumes of DNA Binder Buffer to each DNA sample (200 μL). | |||
**Place mixture in spin column into 2 ml collection tube. | |||
**Centrifuge at full speed for 30 seconds. | |||
**Add 20 μL of DNA Wash Buffer and centrifuge for 30 seconds. Repeat this step. | |||
**Place Zymo-Spin Column into a new 1.5 ml tube. Add 20 μL of water directly to column matrix and spin to elute DNA. | |||
''Gel Electrophoresis'' | |||
* Follow steps for gel electrophoresis. | |||
* Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray. | |||
* Fill gel flask with up to 60 ml of TA buffer. | |||
* Create 1% gel by putting .6 grams of agarose into flask. | |||
* Microwave agarose solution for 40 seconds | |||
* Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 40 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 thick "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. | |||
* Pour gel into tray. | |||
* Wash the agarose gel flask. | |||
* Run gel for 45 min at 100 V. Check gel under UV light. | |||
[[Image:hiddendragonsofthetemple.jpg|250px|CMV PEG/MgCl<sub>2</sub> Precipitation 9/26/2013]] | |||
Higher molecular mass DNA precipitates at lower PEG concentrations than lower molecular mass DNA, according to [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC342844/ Lis and Schleif 1975, NAR]. I kept DNA from the supernatant and discarded the precipitated DNA. | |||
Therefore, I should see larger bands at 2% PEG and smaller bands at 15% PEG. | |||
Latest revision as of 23:23, 26 September 2017
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September 26, 2013Restriction Digest
PEG/MgCl2 Precipitation
DNA Clean & Concentrator
Gel Electrophoresis
Higher molecular mass DNA precipitates at lower PEG concentrations than lower molecular mass DNA, according to Lis and Schleif 1975, NAR. I kept DNA from the supernatant and discarded the precipitated DNA. Therefore, I should see larger bands at 2% PEG and smaller bands at 15% PEG.
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