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 | * Gibson: PCR parts, complete protocol for Brady's construct | ||
* Golden gate: PCR parts; waiting on BsmBI from NEB | |||
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'''PCR''' | '''PCR''' | ||
# V0120, Gib_BBS F (Gib0001) / Gib_BBP R (Gib0002) | # V0120, Gib_BBS F (Gib0001) / Gib_BBP R (Gib0002) | ||
# pSB1A3, Gib_BBS F / Gib_BBP R | # pSB1A3, Gib_BBS F (Gib0001) / Gib_BBP R (Gib0002) | ||
# hPCD, BBP-hPCD fwd (BL9) / hPCD-BL01 rev (BL7) | # hPCD, BBP-hPCD fwd (BL9) / hPCD-BL01 rev (BL7) | ||
# BL01, hPCD-BL01 fwd (BL8) / BL01-BBS rev (BL10) | # BL01, hPCD-BL01 fwd (BL8) / BL01-BBS rev (BL10) | ||
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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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* 72°C, 3 min. | * 72°C, 3 min. | ||
* 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 --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Sample | |||
| bgcolor=#cfcfcf | OD 260 | |||
| bgcolor=#cfcfcf | 260/ 280 | |||
| bgcolor=#cfcfcf | ng/μL | |||
|- | |||
| G1 V0120 || 0.108 || 1.801 || 108.375 | |||
|- | |||
| G2 pSB1A3 || 0.101 || 1.849 || 100.553 | |||
|- | |||
| G3 hPCD || 0.034 || 1.876 || 33.586 | |||
|- | |||
| G4 BL01 || 0.113 || 1.828 || 112.79 | |||
|} | |||
'''Reaction set-up''' | |||
* 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 | |||
* Total volume of DNA = 5.0 μL max | |||
1. hPCD + BL01 + V0120 | |||
* vector V0120, 0.1 pmol = 208 ng = 1.9 μL (use 1.4 μL) | |||
* 2:1 insert hPCD, 0.2 pmol = 24 ng = 0.7 μL | |||
* 2:1 insert BL01, 0.2 pmol = 328 ng = 2.9 μL | |||
2. hPCD + BL01 + V0120 | |||
* vector pSB1A3, 0.1 pmol = 130 ng = 1.3 μL | |||
* 2:1 insert hPCD, 0.2 pmol = 24 ng = 0.7 μL (use 0.8 μL) | |||
* 2:1 insert BL01, 0.2 pmol = 328 ng = 2.9 μL | |||
Gibson reactions: | |||
# hPCD + BL01 + V0120 | |||
# V0120 | |||
# hPCD + BL01 + pSB1A3 | |||
# pSB1A3 | |||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| 1 || 2 || 3 || 4 | |||
|- | |||
| Vector || 1.4 || 1.4 || 1.3 || 1.3 | |||
|- | |||
| Ins 1 || 0.7 || --- || 0.8 || --- | |||
|- | |||
| Ins 2 || 2.9 || --- || 2.9 || --- | |||
|- | |||
| dH<sub>2</sub>O || --- || 3.6 || --- || 3.7 | |||
|- | |||
| || 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 | |||
* Thermal cycler: 50°C,60 min.; 4°C, ∞ | |||
'''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''' | ||
# V0120, gg_BBS F / gg_BBP R | # V0120, gg_BBS F (gg0001) / gg_BBP R (gg0002) | ||
# pSB1A3, gg_BBS F / gg_BBP R | # pSB1A3, gg_BBS F (gg0001) / gg_BBP R (gg0002) | ||
# hPCD, gg_BBP-hPCD F | # hPCD, gg_BBP-hPCD F (gg0003) / gg_hPCD-BL01 R (gg0004) | ||
# BL01, gg_BL01 F / gg_BL01-BBS R | # BL01, gg_BL01 F (gg0004) / gg_BL01-BBS R (gg0005) | ||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | ||
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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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* 72°C, 3 min. | * 72°C, 3 min. | ||
* 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 --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Sample | |||
| bgcolor=#cfcfcf | OD 260 | |||
| bgcolor=#cfcfcf | 260/ 280 | |||
| bgcolor=#cfcfcf | ng/μL | |||
|- | |||
| gg1 V0120 || 0.099 || 1.834 || 98.549 | |||
|- | |||
| gg2 pSB1A3 || 0.068 || 1.758 || 67.725 | |||
|- | |||
| gg3 hPCD || 0.022 || 1.72 || 22.485 | |||
|- | |||
| gg4 BL01 || 0.056 || 1.707 || 55.883 | |||
|} | |||
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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