User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/28: Difference between revisions
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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: | | 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] | ||
|- | |- | ||
| DNA (plasmid) || 25.0 || 15.0 | | DNA (plasmid) || 25.0 || 15.0 | ||
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| 10x buffer || 3.0 || 3.0 | | 10x buffer || 3.0 || 3.0 | ||
|- | |- | ||
| | | EcoRI || 1.0 || 1.0 | ||
|- | |- | ||
| enzyme 2 || 1.0 || 1.0 | | enzyme 2 || 1.0 || 1.0 | ||
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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 | ||
|- | |- | ||
| Insert DNA || | | Insert DNA || 2.0 || --- | ||
|- | |- | ||
| Vector DNA || | | Vector DNA || 2.0 || 2.0 | ||
|- | |- | ||
| 2x lgn buf (Roche) || | | 2x lgn buf (Roche) || 5.0 || 5.0 | ||
|- | |- | ||
| 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 | ||
|- | |- | ||
| || | | || 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] | ||
|- | |- | ||
| 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 | |||
Revision as of 13:24, 29 May 2015
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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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