Difference between revisions of "Part:BBa K1777003:Design"
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WooYehphan (Talk | contribs) (→Overlapping Extension PCR) |
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====Overlapping Extension PCR==== | ====Overlapping Extension 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. <br> | 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. <br> | ||
| − | 1.Use proofreading enzyme for extension. <br> | + | '''1.'''Use proofreading enzyme for extension. <br> |
| − | 2.Run 10-15 PCR cycles without end primers. (Template extension step) <br> | + | '''2.'''Run 10-15 PCR cycles without end primers. (Template extension step) <br> |
| − | 3.Add end primers, then continue cycling for another 15-20 rounds. <br> | + | '''3.'''Add end primers, then continue cycling for another 15-20 rounds. <br> |
| − | 4.Gel extract the correct fragment. Clone into a vector. <br> | + | '''4.'''Gel extract the correct fragment. Clone into a vector. <br><br> |
| − | Tips for recycling:<br> | + | '''Tips for recycling:'''<br> |
| − | Principle: low temperature combination, high temperature elution.<br> | + | *Principle: low temperature combination, high temperature elution.<br> |
| − | Theory: energy will be released when DNA combines with the column and form chemical bond, low temperature is helpful for bond forming. High temperature is good for entropy increase, so DNA fragments is easy to be separated from the column and recycled.<br> | + | *Theory: energy will be released when DNA combines with the column and form chemical bond, low temperature is helpful for bond forming. High temperature is good for entropy increase, so DNA fragments is easy to be separated from the column and recycled.<br> |
===Source=== | ===Source=== | ||
Latest revision as of 13:25, 20 September 2015
miR-21 linear sponge
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 81
Illegal XhoI site found at 452 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 423
Design Notes
6 binding sites are considered to be the most efficient amount of binding site to regulate miR-21. The spacer between the miR-21 binding sites should be designed to prevent mRNA from forming the secondary structure. We got the parts from iDT synthesis DNA fragments, which were made into two parts BBa_K1777001 and BBa_K1777002, and linked them together by overlap extension PCR.
Overlapping Extension 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.
Tips for recycling:
- Principle: low temperature combination, high temperature elution.
- Theory: energy will be released when DNA combines with the column and form chemical bond, low temperature is helpful for bond forming. High temperature is good for entropy increase, so DNA fragments is easy to be separated from the column and recycled.
Source
We use BBa_K1777001 and BBa_K1777002.