Difference between revisions of "Part:BBa K4344048"

Latest revision as of 15:36, 12 October 2022

pUC19-BamHI-fwd.

Part of our primer set used for amplification of pUC19-p19 with: BBa_K4344035 (p19-BamHI-rev.), BBa_K4344036 (p19-ClaI-fwd.), BBa_K4344048 (pUC19-BamHI-fwd.), BBa_K4344049 (pUC19-ClaI-rev.)

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 4
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4344048 short</partinfo>
-p
+Part of our primer set used for amplification of pUC19-p19 with: BBa_K4344035 (p19-BamHI-rev.), BBa_K4344036 (p19-ClaI-fwd.), BBa_K4344048 (pUC19-BamHI-fwd.), BBa_K4344049 (pUC19-ClaI-rev.)
 <!-- Add more about the biology of this part here
 ===Usage and Biology===
+Primers were ordered at IDT. T<sub>m</sub> describes the calculated melting temperature by IDT. T<sub>A</sub> describes the annealing temperature used in the PCR, calculated with NEB T<sub>m</sub> Calculator.
+pUC19-<i>p19</i>-siRNA empty was engineered by restriction based cloning of a PCR amplified backbone and an insert obtained by solid phase synthesis. Primers for pUC19 backbone amplification were analysed on a temperature gradient ranging from 64 °C to 71 °C. The annealing temperature proposed by <a href=”https://tmcalculator.neb.com/#!/main”>NEB T<sub>m</sub>- Calculator</a> was 72 °C. For all temperatures we obtained amplicons with a size ranging from  1500 to 2000 bp as well as a second fragment between 600 and 700 bp . An annealing temperature of 69 °C was chosen and the PCR was repeated. Plasmids received by plasmid preparation were sequenced with <i>p19</i>-forward and AmpR reverse. Insert presence was proven as well as integrity of the loop structure.
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