Haynes Lab:Notebook/Engineering PC-TFs/2013/03/18: Difference between revisions
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== | ==3/18/22== | ||
Cutting inserts to be placed in mammalian vector | Cutting inserts to be placed in mammalian vector | ||
''' A) Get biobricks and enzymes from freezer.''' | ''' A) Get biobricks and enzymes from freezer.''' | ||
PstI, XbaI | |||
The following list of parts are: | The following list of parts are: | ||
* 1. BL01 | |||
* 2. BL05 | |||
* BL01 | * 3. BL09 | ||
* BL05 | * 4. BL12 | ||
* BL09 | |||
* BL12 | |||
{| {{table}} | {| {{table}} | ||
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<font size=3>''B)Follow digest procedure:'' | <font size=3>''B)Follow digest procedure:'' | ||
*DNA 20.0 µL | |||
*Enzyme 1 1.0 µL | |||
*Enzyme 2 1.0 µL | |||
*10x buffer 3.0 µL | |||
*dH2O 5.0 uL | |||
* = 30.0 uL </font> | |||
|} | |} | ||
''Digest Order:'' | ''Digest Order:'' | ||
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*4 BL12 XbaI/PstI | *4 BL12 XbaI/PstI | ||
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:gel3_22_2013.jpg|200px|Stock digest 3/ | {| {{table}} cellspacing="3" <!-- Digest rxn. table --> | ||
[[Image:gel3_22_2013_cutout.jpg|200px|Stock digest 3/ | |- valign="top" | ||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Volume | |||
| rowspan="7" | [[Image:gel3_22_2013.jpg|200px|Stock digest 3/18/2013]] [[Image:gel3_22_2013_cutout.jpg|200px|Stock digest 3/18/2013]] <br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|- | |||
| DNA (plasmid) || 15.0 μL | |||
|- | |||
| XbaI|| 1.0 μL | |||
|- | |||
| PstI|| 1.0 μL | |||
|- | |||
| Green 10x FastDigest buffer || 1.0 μL | |||
|- | |||
| dH<sub>2</sub>O || 10 μL | |||
|- | |||
| || 30 μL --> 37°C/ ~10 min. | |||
|} | |||
{|border="1" cellpadding="5" cellspacing="0" align=" | {|border="1" cellpadding="5" cellspacing="0" align="left" | ||
|- | |- | ||
! scope="col" style="background:#efefef;" | # | ! scope="col" style="background:#efefef;" | # |
Revision as of 20:34, 22 March 2013
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3/18/22Cutting inserts to be placed in mammalian vector A) Get biobricks and enzymes from freezer. PstI, XbaI The following list of parts are:
Digest Order: Biobrick Restriction Enzymes
Follow steps for gel electrophoresis.
|