Difference between revisions of "Part:BBa K747100"
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<partinfo>BBa_K747100 short</partinfo> | <partinfo>BBa_K747100 short</partinfo> | ||
| − | + | Precise Gene Knockout | |
TALENs are a very powerful tool for efficient gene knockout. In order to prove that our TALEN construct was functional, we decided to simply knock out a destabilized GFP gene on a plasmid, which we co-transfected with our TALEN plasmids into HEK cells. Moreover, we also transfected our cells with an mCherry vector to normalize for transfection efficiency. TAL constructs were designed to bind to opposite strands of the target plasmid in a way that the FokI monomers of each TALEN construct would be able to dimerize in the spacer region between the TALEN binding sites. 48 hours after transfection, gene knock-out efficiency was evaluated by FACS analysis. | TALENs are a very powerful tool for efficient gene knockout. In order to prove that our TALEN construct was functional, we decided to simply knock out a destabilized GFP gene on a plasmid, which we co-transfected with our TALEN plasmids into HEK cells. Moreover, we also transfected our cells with an mCherry vector to normalize for transfection efficiency. TAL constructs were designed to bind to opposite strands of the target plasmid in a way that the FokI monomers of each TALEN construct would be able to dimerize in the spacer region between the TALEN binding sites. 48 hours after transfection, gene knock-out efficiency was evaluated by FACS analysis. | ||
Revision as of 15:46, 28 October 2012
pTALEN
Precise Gene Knockout
TALENs are a very powerful tool for efficient gene knockout. In order to prove that our TALEN construct was functional, we decided to simply knock out a destabilized GFP gene on a plasmid, which we co-transfected with our TALEN plasmids into HEK cells. Moreover, we also transfected our cells with an mCherry vector to normalize for transfection efficiency. TAL constructs were designed to bind to opposite strands of the target plasmid in a way that the FokI monomers of each TALEN construct would be able to dimerize in the spacer region between the TALEN binding sites. 48 hours after transfection, gene knock-out efficiency was evaluated by FACS analysis.
Sequence and Features
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 966
Illegal suffix found in sequence at 4462 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 966
Illegal SpeI site found at 4463
Illegal PstI site found at 4477
Illegal NotI site found at 972
Illegal NotI site found at 2146
Illegal NotI site found at 4470 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 966
Illegal BglII site found at 1563
Illegal BglII site found at 2914
Illegal BamHI site found at 2217
Illegal BamHI site found at 2920 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 966
Illegal suffix found in sequence at 4463 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 966
Illegal XbaI site found at 981
Illegal SpeI site found at 4463
Illegal PstI site found at 4477 - 1000COMPATIBLE WITH RFC[1000]