User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/17: 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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==04/ | ==04/17/15== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* Rene - gRNA plasmids, 5 mL cultures (11 total) | * Rene - gRNA plasmids, 5 mL cultures (11 total) | ||
* Ryan - Receiver plasmids, assembly | * Ryan - Receiver plasmids, assembly | ||
* LCR Development - Repeat Phusion for MV10, use correct amount of 5x buffer | |||
---- | ---- | ||
'''Ryan - Receiver plasmids, assembly''' | '''Ryan - Receiver plasmids, assembly''' | ||
* Stage 1 - insert Regulator ORFS | * Stage 1 - insert Regulator ORFS | ||
** Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites) | ** Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites) | ||
** Insert ORF(E/X) into Vector ( | ** Insert ORF(E/X) into Vector (E/X). see [https://benchling.com/s/VFNzuauP/edit Vector plasmid map] | ||
* Stage 2 - insert Promoters | * Stage 2 - insert Promoters | ||
** Cut & dephos promoters with E/X | ** Cut & dephos promoters with E/X | ||
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Phusion PCR | Phusion PCR | ||
# | # AubR, EcoRI F/ XbaI R | ||
# | # BjaR, " | ||
# | # BraR, " | ||
# | # RpaR, " | ||
{| {{table}} border="1" cellspacing="3" <!-- Phusion PCR table --> | {| {{table}} border="1" cellspacing="3" <!-- Phusion PCR table --> | ||
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| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| bgcolor=#cfcfcf | Mix ( | | bgcolor=#cfcfcf | Mix (x5) | ||
| rowspan="7" | <u>Expected:</u><br>1. | | rowspan="7" | <u>Expected:</u><br>1. AubR = 851<br>2. BjaR = 776<br>3. BraR = 776<br>4. RpaR = 779<br> | ||
| rowspan="7" | | | rowspan="7" | [[Image:KAH041715_gel1.jpg|200px|Hover name]]<br>5 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 || --- | ||
|- | |- | ||
| 10 μM F primer || 1.0 || | | 10 μM F primer || 1.0 || 5.0 | ||
|- | |- | ||
| 10 μM R primer || 1.0 || | | 10 μM R primer || 1.0 || 5.0 | ||
|- | |- | ||
| 10 mM dNTPs || 1.0 || | | 10 mM dNTPs || 1.0 || 5.0 | ||
|- | |- | ||
| Phusion Pol. || 0.5 || | | Phusion Pol. || 0.5 || 2.5 | ||
|- | |- | ||
| 5X HF buffer || 10.0 || | | 5X HF buffer || 10.0 || 50.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || 36.0 || | | dH<sub>2</sub>O || 36.0 || 180.0 | ||
|- | |- | ||
| || 50.0 || | | || 50.0 || | ||
|} | |} | ||
Program: Phusion (block B) | |||
* 98°C, 3 min | |||
* 35x[98°C, 10 sec; 66°C, 30 sec; 72°C, 60 sec] | |||
* 72°C, 10 min | |||
* 4°C ∞ | |||
Conclusion | |||
* All four worked. Proceed to next step | |||
---- | ---- | ||
''' | '''LCR Development''' | ||
* Repeat Phusion for MV10 (from 4/16/15), use correct amount of 5x buffer | |||
Phusion PCR | |||
{| {{table}} cellspacing="3" <!-- | {| {{table}} border="1" cellspacing="3" <!-- Phusion PCR table --> | ||
|- valign="top" | |- valign="top" | ||
| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | | | bgcolor=#cfcfcf | Rxn1,2 | ||
| rowspan="7" | < | | rowspan="7" | <u>Expected:</u><br>1,2. MV10 = 5191 | ||
| rowspan="7" | [[Image:KAH041715_gel2.jpg|150px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|- | |- | ||
| | | DNA(clean linear) || 1.0 μL | ||
|- | |- | ||
| | | 10 μM F primer || 1.0 | ||
|- | |- | ||
| | | 10 μM R primer || 1.0 | ||
|- | |- | ||
| 1. | | 10 mM dNTPs || 1.0 | ||
|- | |- | ||
| | | Phusion Pol. || 0.5 | ||
|- | |- | ||
| | | 5X HF buffer || 10.0 | ||
|- | |- | ||
| | | dH<sub>2</sub>O || 35.5 | ||
|- | |- | ||
| | | || 50.0 || | ||
|} | |} | ||
Program: Phusion (block A) | |||
* 98°C, 3 min | |||
* 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 2.5 min] | |||
* 72°C, 10 min | |||
* 4°C ∞ | |||
Conclusion | |||
* Big improvement. Keep this for potential cloning | |||
* Also try again with higher annealing temp | |||
Latest revision as of 00:55, 27 September 2017
Karmella's BioBrick Cloning | Main project page Previous entry Next entry | ||||||||||||||||||||||||||||||||||||||||||||||||||
04/17/15
Ryan - Receiver plasmids, assembly
Conclusion
LCR Development
Conclusion
|