User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/28: Difference between revisions
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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 | |||
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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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