Haynes Lab:Notebook/Engineering PC-TFs/2013/10/05: Difference between revisions
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== | ==October 5, 2013== | ||
* | * PEG/MgCl<sub>2</sub> | ||
** Make 50 mL | |||
***Put water, then MgCl<sub>2</sub>, then PEG 8000 in 50 mL conical tube | |||
{|border="1" cellpadding="6" cellspacing="0" align="left" | |||
|- | |||
! scope="col" style="background:#efefef;" | Reagent | |||
! scope="col" style="background:#efefef;" | Volume | |||
|- | |||
|PEG 8000 | |||
|15 g/mL | |||
|- | |||
|MgCl<sub>2</sub> | |||
|1.5 mL | |||
|- | |||
|H<sub>2</sub>O | |||
|33.5 mL | |||
|- | |||
|'''Total''' | |||
|50 mL | |||
|- | |||
|} | |||
<br> <br> <br> <br> <br> <br> <br> <br> <br> <br> <br> | |||
*Perform restriction digest for CMV promoter | |||
{| {{table}} cellspacing="3" <!-- Digest rxn. table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | '''Reagent''' | |||
| bgcolor=#cfcfcf | '''Volume''' | |||
|- | |||
| DNA (plasmid) || 10 μL | |||
|- | |||
| XbaI|| 1.0 μL | |||
|- | |||
| PstI|| 1.0 μL | |||
|- | |||
| 10x FastDigest buffer || 3μL | |||
|- | |||
| dH<sub>2</sub>O || 15 μL | |||
|- | |||
| || 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. | |||
{| {{table}} cellspacing="3" <!-- Digest rxn. table --> | |||
| bgcolor=#cfcfcf | '''Reagent''' | |||
| bgcolor=#cfcfcf | '''15%''' | |||
| bgcolor=#cfcfcf | '''10%''' | |||
| bgcolor=#cfcfcf | '''7%''' | |||
| bgcolor=#cfcfcf | '''5%''' | |||
| bgcolor=#cfcfcf | '''2%''' | |||
|- | |||
| 30% PEG || 50 μL|| 33 μL || 23 μL|| 17 μL || 7 μL | |||
|- | |||
| DNA|| 15μL||15 μL||15 μL||15 μL||15 μL | |||
|- | |||
| dH<sub>2</sub>O|| 35 μL||52 μL||62 μL||68 μL||78 μL | |||
|- | |||
| Total || 100 μL || 100 μL|| 100 μL|| 100 μL|| 100 μL | |||
|} | |||
*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:ihavenoideawhyitsnotworking.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. | |||
Revision as of 21:06, 11 October 2013
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October 5, 2013
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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