Difference between revisions of "Cloning human alpha casein S1, 04Oct2014 and on"
Jump to navigation
Jump to search
m (→Overview) |
|||
Line 8: | Line 8: | ||
*'''Participants:''' Meenakshi, Johan, Lafia | *'''Participants:''' Meenakshi, Johan, Lafia | ||
*'''Notes:''' | *'''Notes:''' Lafia, transcribed by Rachel | ||
*'''Location:''' BioCurious | *'''Location:''' BioCurious | ||
*'''Materials & Methods, Electra Cloning:''' | |||
**We resuspended lyophilized IDT gBlock DNA for P.FAKS.humAlphaS1.S, received 04Oct2014, as in [[DNA Handling]] using 0.5X TE (1X TE, pH 7.5: 1 diH2O). Final DNA concentration = 20ng/uL, per Electra cloning kit requirements. | |||
** Used [https://www.dna20.com/products/expression-vectors/electra-system#2 DNA 2.0 Electra cloning kit] protocol. | |||
Reaction: | |||
1uL pD1214 (20ng) | |||
1uL of appropriate casein gBlock or (+) control DNA (20ng), or 0.5X TE (-) control | |||
2uL Electra Buffer | |||
1uL of Electra enzyme mix | |||
15uL of diH20 | |||
---- | |||
20uL | |||
Incubated 20 min room temperature | |||
== Transforming human alpha casein S1:pD1214 cloning reaction product into ''E. coli''== | == Transforming human alpha casein S1:pD1214 cloning reaction product into ''E. coli''== |
Revision as of 07:19, 16 October 2014
Cloning human alpha casein S1, plasmid DNA midiprep and DNA conc./purity
Overview
During these experiments, we will insert our final IDT gBlock, human alpha casein (received 03Oct2014,) into the pD1214 vector and transform our constructs into E. coli. We will then perform midiprep plasmid DNA extractions, quantify our plasmid DNA and submit plasmid DNA from duplicate clones for sequencing. Sequence-confirmed DNA is then ready to be transformed into yeast.
Cloning casein & FAM20C gBlocks into pD1214
October 4, 2014
- Participants: Meenakshi, Johan, Lafia
- Notes: Lafia, transcribed by Rachel
- Location: BioCurious
- Materials & Methods, Electra Cloning:
- We resuspended lyophilized IDT gBlock DNA for P.FAKS.humAlphaS1.S, received 04Oct2014, as in DNA Handling using 0.5X TE (1X TE, pH 7.5: 1 diH2O). Final DNA concentration = 20ng/uL, per Electra cloning kit requirements.
- Used DNA 2.0 Electra cloning kit protocol.
Reaction:
1uL pD1214 (20ng) 1uL of appropriate casein gBlock or (+) control DNA (20ng), or 0.5X TE (-) control 2uL Electra Buffer 1uL of Electra enzyme mix 15uL of diH20 ---- 20uL Incubated 20 min room temperature
Transforming human alpha casein S1:pD1214 cloning reaction product into E. coli
October 4, 2014
- Participants: Lafia, Meenakshi, Johan
- Notes:
- Location: BioCurious