User:Karmella Haynes/Notebook/BioBrick cloning/2013/08/29: Difference between revisions
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==08/29/13== | ==08/29/13== | ||
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* Golden Gate Assembly optimization - increase ligase amount; redo hPCD assembly | * Golden Gate Assembly optimization - increase ligase amount; redo last successful hPCD assembly | ||
---- | ---- | ||
'''Golden Gate''' | '''Golden Gate''' | ||
* Try to increase robustness by using more T4 ligase | * Try to increase robustness by using more T4 ligase... | ||
# hPCD + BL01 + pSB1A3 | # hPCD + BL01 + pSB1A3 | ||
# pSB1A3 alone (neg ctrl) | # pSB1A3 alone (neg ctrl) | ||
* GG-PCR fragments were generated on [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/01/10 1/10/13] and diluted to 20 fmol/μL | * GG-PCR fragments were generated on [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/01/10 1/10/13] and diluted to 20 fmol/μL | ||
* Try the Promega formula home-made buffer (worked last time) | |||
* Golden Gate assembly reactions | |||
1, 2. 0.25 μL T4 ligase, hPCD+BL01+pSB1A3 or pSB1A3<br> | |||
1, 2. 1.0 μL T4 ligase, hPCD+BL01+pSB1A3 or pSB1A3<br> | |||
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table --> | {| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table --> | ||
|- valign="top" | |- valign="top" | ||
| Reagent || | | Reagent || Original protocol || Original protocol || More ligase || More ligase || H2B+LOV | ||
|- | |- | ||
| gg2 pSB1A3 || 1.0 || 1.0 | | gg2 pSB1A3* || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 | ||
|- | |- | ||
| gg3 hPCD || 1.0 || --- | | gg3 hPCD* || 1.0 || --- || 1.0 || --- || 1.0 H2B gg | ||
|- | |- | ||
| gg4 BL01 || 1.0 || --- | | gg4 BL01* || 1.0 || --- || 1.0 || --- || 1.0 LOV gg | ||
|- | |- | ||
| 10x ligase buffer || 1.0 || 1.0 | | 10x Promega ligase buffer || 1.0 || 1.0|| 1.0 || 1.0 || 1.0 | ||
|- | |- | ||
| NEB T4 | | NEB T4 ligase || 0.25 || 0.25 || 1.0 || 1.0 || 1.0 | ||
|- | |- | ||
| BsmBI || 0.5 || 0.5 | | NEB BsmBI || 0.5 || 0.5 || 0.5 || 0.5 || 0.5 | ||
|- | |- | ||
| dH<sub>2</sub>O || 5.25 || 7.25 | | dH<sub>2</sub>O || 5.25 || 7.25 || 4.5 || 6.5 || 4.5 | ||
|- | |- | ||
| || 10.0 || 10.0 | | || 10.0 || 10.0 || 10.0 || 10.0 || 10.0 | ||
|} | |} | ||
Note: *DNA is 20 fmol/μL | |||
GG Thermal cycling | GG Thermal cycling | ||
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* Transformations (08/30/13) | |||
** 40 μL '''BL21''' in 2.0 mL tubes; ice 2 min.; 42°C 45 sec.; add 750 μL SOC medium; shake @ 37°C 30 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar | |||
* Results | |||
** No colonies on any plates. Start again from generation of PCR fragments. Try DpnI clean-up approach next time. | |||
Revision as of 12:09, 31 August 2013
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08/29/13
Golden Gate
1, 2. 0.25 μL T4 ligase, hPCD+BL01+pSB1A3 or pSB1A3
Note: *DNA is 20 fmol/μL GG Thermal cycling
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