User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/28: 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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| bgcolor=#cfcfcf | Rxn1 | | bgcolor=#cfcfcf | Rxn1 | ||
| bgcolor=#cfcfcf | Rxn2-5 | | bgcolor=#cfcfcf | Rxn2-5 | ||
| rowspan="7" | Expected bands:<br>1. hPCD(E/S) = 186<br>2. PLflex_pUC57(E/X) = 2763<br>3. PLflex4_pUC57(E/X) = 2930<br>4. PLrigid_pUC57(E/X) = 2763<br>5. PLrigid4_pUC57(E/X) = 2931 | | rowspan="7" | Expected bands:<br>1. hPCD(E/S) = 3200, 186*<br>2. PLflex_pUC57(E/X) = 2763*<br>3. PLflex4_pUC57(E/X) = 2930*<br>4. PLrigid_pUC57(E/X) = 2763*<br>5. PLrigid4_pUC57(E/X) = 2931*<br><br>(*) Bands that were cut out for purification | ||
| rowspan="7" | [[Image:KAH052815_gel1.jpg|250px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | | rowspan="7" | [[Image:KAH052815_gel1.jpg|250px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
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| 5. PLrigid4_pUC57(E/X) || --- || --- || --- | | 5. PLrigid4_pUC57(E/X) || --- || --- || --- | ||
|} | |} | ||
** Readings are very odd, which is typical for the Sigma gel extraction products. | |||
** Will try my luck with these and use an arbitrary volume of 2.0 insert and 2.0 vector. | |||
** Good insert : vector ratio should not be difficult to achieve since insert is very short. | |||
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# DT004_pUC57: hPCD(E/S)/186 + PLrigid4_pUC57(E/X)/2931 | # DT004_pUC57: hPCD(E/S)/186 + PLrigid4_pUC57(E/X)/2931 | ||
# PLflex_pUC57(E/X)/2763 | # PLflex_pUC57(E/X)/2763 | ||
* Note: not enough plates to do neg. ctrls for all vectors. Used just one of the vectors | |||
{| {{table}} cellspacing="3" <!-- Ligation rxn table --> | {| {{table}} cellspacing="3" <!-- Ligation rxn table --> | ||
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| bgcolor=#cfcfcf | Rxn5 | | bgcolor=#cfcfcf | Rxn5 | ||
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| Insert DNA || | | Insert DNA || 2.0 || --- | ||
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| Vector DNA || | | Vector DNA || 2.0 || 2.0 | ||
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| 2x lgn buf (Roche) || | | 2x lgn buf (Roche) || 5.0 || 5.0 | ||
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| T4 ligase (NEB) || 1.0 || 1.0 | | T4 ligase (NEB) || 1.0 || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || --- || 2.0 | ||
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| || | | || 10.0 μL || 10.0 μL | ||
|} | |} | ||
RESULTS | |||
* Looks successful - David Tze: ~200 colonies on the ligation plates, 1 colony on the neg. ctrl plate. | |||
* David Tze: picked two colonies from each plate (1-4) for streak library and 5 mL cultures | |||
---- | ---- | ||
''' | '''Ben - DBN007 5 mL cultures''' | ||
* DBN001_psB1A3: https://benchling.com/s/S2BS6ziD/edit | * DBN001_psB1A3: https://benchling.com/s/S2BS6ziD/edit | ||
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| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| rowspan="7" | <u>Expected:</u><br>1-2. DBN007_pSB1A3 = 2197, 2132<br>DBN001 = 2132, 224<br> | | rowspan="7" | <u>Expected:</u><br>1-2. DBN007_pSB1A3 = 2197, 2132<br>DBN001 = 2132, 224<br> | ||
| rowspan="7" | [[Image: | | rowspan="7" | [[Image:KAH052815_gel2.jpg|150px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
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| DNA(plasmid) || 2.0 μL | | DNA(plasmid) || 2.0 μL | ||
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CONCLUSIONS | |||
* Looks successful. 2197, 2132 bands are hard to resolve at the 2000 kb range. | |||
* Submit the plasmids for sequencing | |||
Latest revision as of 00:59, 27 September 2017
Karmella's BioBrick Cloning | Main project page Previous entry Next entry | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
05/28/15
David Tze - assemblies: linkers + hPCD
--> 37°C/ ~30 min.
Ben - DBN007 5 mL cultures
--> 37°C/ ~15 min.
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