User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/05: Difference between revisions
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==01/05/13== | ==01/05/13== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* | * Gibson: PCR parts, complete protocol for Brady's construct | ||
* | * Golden gate: PCR parts; waiting on BsmBI from NEB | ||
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| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| rowspan="7" | <u>Expected:</u><br> | | rowspan="7" | <u>Expected:</u><br>G1. V0120 = 3200<br>G2. pSB1A3 = 2000<br>G3. hPCD = 186<br>G4. BL01 = 2520 | ||
| rowspan="7" | [[Image: | | rowspan="7" | [[Image:KAH010513_gel1.jpg|300px|PCR gel]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
|- | |- | ||
| DNA(plasmid) || 0.5 μL | | DNA(plasmid) || 0.5 μL | ||
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* 4°C, ∞ | * 4°C, ∞ | ||
''' | |||
'''Purification''' | |||
* Cleaned remaining 40 μL of PCR using the Zymo DNA Clean & Concentrator kit | |||
* Eluted samples with 20 μL dH<sub>2</sub>O | |||
'''DNA Concentrations''' | |||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | ||
|- valign="top" | |- valign="top" | ||
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'''Reaction set-up''' | '''Reaction set-up''' | ||
* Use 0.2 - 0.5 pmol, per NEB's suggestion | * Use 0.2 - 0.5 pmol, per NEB's suggestion, [http://www.neb.com/nebecomm/products/protocol819.asp] | ||
* pmols = ng x 1000 / bp x 650 daltons; ng = (pmols x bp x 650 daltons) / 1000 | * pmols = ng x 1000 / bp x 650 daltons; ng = (pmols x bp x 650 daltons) / 1000 | ||
* Total volume of DNA = 5.0 μL max | * Total volume of DNA = 5.0 μL max | ||
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| dH<sub>2</sub>O || --- || 3.6 || --- || 3.7 | | dH<sub>2</sub>O || --- || 3.6 || --- || 3.7 | ||
|- | |- | ||
| || 5.0 μL | | || 5.0 μL || 5.0 μL || 5.0 μL || 5.0 μL | ||
|} | |} | ||
* Add each to one aliquot of 15.0 Gibson mix (Rene); total = 20.0 μL | * Add each to one aliquot of 15.0 Gibson mix (Rene); total = 20.0 μL | ||
* Thermal cycler: 50°C,60 min.; 4°C, ∞ | * Thermal cycler: 50°C,60 min.; 4°C, ∞ | ||
* Transform | |||
'''Transformation''' | |||
* (1) Transform: add '''2.0 μL''' to 40 μL DH5α-Turbo; ice/5 min.; plate on warm 100 μg/mL amp. agar | |||
* (2) NEB also suggests diluting 5.0 μL of the Gibson reaction into 15.0 μL dH<sub>2</sub>O (20.0 total vol.), and using 2.0 μL diluted DNA, [http://www.neb.com/nebecomm/products/protocol820.asp] | |||
* Test both methods (8 plates total) | |||
** Plates 1-4, method 1 | |||
** Plates 5-8, method 2 | |||
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# hPCD + BL01 + pSB1A3 | # hPCD + BL01 + pSB1A3 | ||
* Check the sequences for BsmBI sites | |||
* Check the sequences for BsmBI docking sites (CGTCTC) | |||
** hPCD - none | |||
** BL01: hPCD-mCherry-SP1AB - none | |||
** V0120 - <font color="red">5 sites</font> | |||
** pSB1A3 - none | |||
'''PCR''' | '''PCR''' | ||
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| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| rowspan="7" | <u>Expected:</u><br> | | rowspan="7" | <u>Expected:</u><br>gg1. V0120 = 3200<br>gg2. pSB1A3 = 2000<br>gg3. hPCD = 186<br>gg4. BL01 = 2520 | ||
| rowspan="7" | [[Image: | | rowspan="7" | [[Image:KAH010513_gel1.jpg|300px|PCR gel]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
|- | |- | ||
| DNA(plasmid) || 0.5 μL | | DNA(plasmid) || 0.5 μL | ||
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''' | |||
'''Purification''' | |||
* Cleaned remaining 40 μL of PCR using the Zymo DNA Clean & Concentrator kit | |||
* Eluted samples with 20 μL dH<sub>2</sub>O | |||
'''DNA Concentrations''' | |||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | ||
|- valign="top" | |- valign="top" |
Revision as of 19:42, 5 January 2013
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01/05/13
Gibson Assembly
1. hPCD + BL01 + V0120
2. hPCD + BL01 + V0120
Golden Gate Assembly
Purification
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