Difference between revisions of "Part:BBa K2446037:Design"
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<B>OE-PCR:</B> PCR amplify and connecting the necessary fragments, using proofreading polymerase enzyme. They should have about 15-25 bp overlaps. Use oligo Tm calculators to figure out their annealing temperature. Clean up or gel extract the correct size band. Use cleaned up fragments as "template". Unlike normal PCR, about 1/2 to 3/4 volume of the extension reaction should be template. | <B>OE-PCR:</B> PCR amplify and connecting the necessary fragments, using proofreading polymerase enzyme. They should have about 15-25 bp overlaps. Use oligo Tm calculators to figure out their annealing temperature. Clean up or gel extract the correct size band. Use cleaned up fragments as "template". Unlike normal PCR, about 1/2 to 3/4 volume of the extension reaction should be template. | ||
+ | |||
1.Use proofreading enzyme for extension. | 1.Use proofreading enzyme for extension. | ||
+ | |||
2.Run 10-15 PCR cycles without end primers. (Template extension step) | 2.Run 10-15 PCR cycles without end primers. (Template extension step) | ||
+ | |||
3.Add end primers, then continue cycling for another 15-20 rounds. | 3.Add end primers, then continue cycling for another 15-20 rounds. | ||
+ | |||
4.Gel extract the correct fragment. Clone into a vector. | 4.Gel extract the correct fragment. Clone into a vector. | ||
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===Source=== | ===Source=== | ||
− | + | The three fragments are from IDT. | |
===References=== | ===References=== | ||
+ | [1] Morsut, L. et al. Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell 164, 780--791 (2016). | ||
+ | |||
+ | [2] Witzgall, R., O'Leary, E., Leaf, A., Onaldi, D. & Bonventre, J. V. The Krüppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression. Proceedings of the National Academy of Sciences 91, 4514-4518 (1994). | ||
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+ | [3] Chen, X., Zaro, J. L. & Shen, W.-C. Fusion protein linkers: property, design and functionality. Advanced drug delivery reviews 65, 1357-1369 (2013). |
Latest revision as of 01:13, 30 October 2017
ZF_GAl4_KRAB
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 256
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 256
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 256
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 256
Illegal NgoMIV site found at 41
Illegal NgoMIV site found at 176 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
This part was a fusion protein which assembled by three fragments OE-PCR. It contain three core domains from N-terminal to C-terminal: GAL4 DNA binding domain (DBD), nuclear location sequence and transcription regulating domain.
OE-PCR: PCR amplify and connecting the necessary fragments, using proofreading polymerase enzyme. They should have about 15-25 bp overlaps. Use oligo Tm calculators to figure out their annealing temperature. Clean up or gel extract the correct size band. Use cleaned up fragments as "template". Unlike normal PCR, about 1/2 to 3/4 volume of the extension reaction should be template.
1.Use proofreading enzyme for extension.
2.Run 10-15 PCR cycles without end primers. (Template extension step)
3.Add end primers, then continue cycling for another 15-20 rounds.
4.Gel extract the correct fragment. Clone into a vector.
Source
The three fragments are from IDT.
References
[1] Morsut, L. et al. Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell 164, 780--791 (2016).
[2] Witzgall, R., O'Leary, E., Leaf, A., Onaldi, D. & Bonventre, J. V. The Krüppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression. Proceedings of the National Academy of Sciences 91, 4514-4518 (1994).
[3] Chen, X., Zaro, J. L. & Shen, W.-C. Fusion protein linkers: property, design and functionality. Advanced drug delivery reviews 65, 1357-1369 (2013).