Haynes Lab:Notebook/Engineering PC-TFs/2013/03/18
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< Haynes Lab:Notebook | Engineering PC-TFs | 2013 | 03(Difference between revisions)
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Current revision (23:34, 22 March 2013) (view source) (→3/18/22) |
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<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
| - | == | + | ==3/18/22== |
Cutting inserts to be placed in mammalian vector | Cutting inserts to be placed in mammalian vector | ||
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''' 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> | |
| - | + | ||
|} | |} | ||
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''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. | ||
{| {{table}} cellspacing="3" <!-- Digest rxn. table --> | {| {{table}} cellspacing="3" <!-- Digest rxn. table --> | ||
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| 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] | | 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) || | + | | 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 || | + | | dH<sub>2</sub>O || 10 μL |
|- | |- | ||
| - | | || 30 μL --> 37°C/ ~ | + | | || 30 μL --> 37°C/ ~10 min. |
|} | |} | ||
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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.
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