Difference between revisions of "Part:BBa K2446037:Design"
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===Design Notes=== | ===Design Notes=== | ||
− | + | <div> | |
− | + | 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. | |
− | + | </div> | |
+ | <div> | ||
+ | <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. | ||
+ | 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. | ||
+ | </div> | ||
===Source=== | ===Source=== |
Revision as of 01:10, 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
waiting