User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/15: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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== | |||
==01/15/13== | |||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* | * Golden gate assembly: test different ligase buffers | ||
---- | ---- | ||
''' | '''Golden Gate Assembly optimization'''<br> | ||
* | * Test different buffers | ||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | ||
|- valign="top" | |- valign="top" bgcolor=#cfcfcf | ||
| Buffer || Tris-HCl || MgCl<sub>2</sub> || DTT || ATP | |||
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| | |||
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|- | |- | ||
| | | 10x Promega || 300 mM (pH 7.8) || 100 mM || 100 mM || 10 mM | ||
|- | |- | ||
| | | 10x NEB || 500 mM (pH 7.5) || 100 mM || 100 mM || 10 mM | ||
|- | |- | ||
| | | 2x Roche || ? || ? || ? || ? | ||
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| | |||
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|} | |} | ||
* Make Promega buffer from scratch. | |||
{| {{table}} cellspacing="3" <!-- | * Golden Gate assembly reactions | ||
1, 2. Promega - hPCD+BL01+pSB1A3<br> | |||
3, 4. Promega - pSB1A3<br> | |||
5, 6. NEB - hPCD+BL01+pSB1A3<br> | |||
7, 8. NEB - pSB1A3<br> | |||
9, 10. Roche - hPCD+BL01+pSB1A3<br> | |||
11, 12. Roche - pSB1A3<br> | |||
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table --> | |||
|- valign="top" | |- valign="top" | ||
| | | Reagent || Promega (1,2) || Promega (3, 4) || NEB (5, 6) || NEB (7, 8) || Roche (9, 10) || Roche (11, 12) | ||
| | |||
| | |||
|- | |- | ||
| | | gg2 pSB1A3* || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 | ||
|- | |- | ||
| | | gg3 hPCD* || 1.0 || --- || 1.0 || --- || 1.0 || --- | ||
|- | |- | ||
| | | gg4 BL01* || 1.0 || --- || 1.0 || --- || 1.0 || --- | ||
|- | |- | ||
| | | ligase buffer || 1.0 || 1.0|| 1.0 || 1.0|| 5.0 || 5.0 | ||
|- | |- | ||
| | | NEB T4 ligase || 0.25 || 0.25 || 0.25 || 0.25 || 0.25 || 0.25 | ||
|- | |- | ||
| | | NEB BsmBI || 0.5 || 0.5 || 0.5 || 0.5 || 0.5 || 0.5 | ||
| | |||
| | |||
| | |||
|- | |- | ||
| | | dH<sub>2</sub>O || 5.25 || 7.25 || 5.25 || 7.25 || 1.25 || 3.25 | ||
|- | |- | ||
| | | || 10.0 || 10.0 || 10.0 || 10.0 || 10.0 || 10.0 | ||
|} | |} | ||
Note: *DNA is 20 fmole/μL | |||
Thermal cycler | |||
* [45°C, 2 min.; 16°C, 5 min.] x25 | |||
* 60°C, 20 min. | |||
* 80°C, 20 min. | |||
* 4°C, ∞ | |||
* Transformations | |||
** All odd samples: 50 μL '''DH5α-Turbo'''; ice 5 min.; plate on amp agar | |||
** All even samples: 50 μL '''BL21''' in 2.0 mL tubes; ice 2 min.; 42°C 90 sec.; add 800 μL SOC medium; shake @ 37°C 25 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar | |||
---- | ---- | ||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> |
Latest revision as of 22:21, 26 September 2017
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01/15/13
Golden Gate Assembly optimization
1, 2. Promega - hPCD+BL01+pSB1A3
Note: *DNA is 20 fmole/μL Thermal cycler
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