Difference between revisions of "Team:Tongji China/Process"

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Revision as of 12:56, 1 November 2017


Tongji iGEM - Process
Tongji iGEM
TongJi iGEM
Process
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Backbone

Plasmid Backbone

Clone pleP, Gal4, Gal80 and TH

Gal4 (2646bp) integrated with UAS-Gal4 system, and pleP (452bp) and Gal80ts (1311bp) are from the genome of Drosophila melanogaster integrated with a mutant Gal80ts. Because of the repetitive sequence on the TH, it is difficult to clone so we order the synthetic pUAST (double digestion with EcoRI and XbaI)-TH from GENEWIZ®.

NO DESCRIPTION

NO DESCRIPTION
NO DESCRIPTION

Digest pUAST-3xHA

For constructing the plasmid pleP-Gal4 and pleP-Gal80ts, avoiding the influence of UAS on the backbone we should remove it. We remove UAS by BamHI digestion.

NO DESCRIPTION
NO DESCRIPTION
A) pUAST-3xHA
B) pUAST-3xHA BamHI (for 6)

Assembly

To assembly the digested backbone and fragment. We take two ways: to insert a new MCS into the backbone (done), then use a method of double digestion to insert two fragments one by one (failed), or to single digest, then use seamless cloning to insert two fragment together (success).

A) To insert a new MCS into the backbone (done), then use a method of double digestion to insert two fragments one by one (failed)
1. Transform backbone with a new MCS

NO DESCRIPTION
NO DESCRIPTION


2. BglII digestion to insert pleP (done)

NO DESCRIPTION
Digestion
A) pUAST-oligo BglII
B) pUAST-oligo
NO DESCRIPTION
pUAST-oligo-pleP


3. pUAST-new MCS-pleP digestion (done) to insert Gal4 and Gal80ts (failed)

NO DESCRIPTION
Digestion
A) pUAST-new MCS -pleP
B) pUAST-new MCS -pleP NotI
C) pUAST-new MCS -pleP HpaI
D) pUAST-new MCS -pleP NotI&HpaI (For 11)


B) To single digest, then use seamless cloning to insert two fragment together (success)
1. BamHI digestion (2 done)
2. Seamless cloning to insert two fragment together (done)
We have done the enzyme digestion to verify them

1% gel electrophoresis of plasmid
pUAST-pleP-Gal4
A) pUAST-pleP-Gal4
B) pUAST-pleP-Gal4 PstI
1% gel electrophoresis of plasmid
pUAST-pleP-Gal80ts
A) pUAST-pleP-Gal80ts
B) pUAST-pleP-Gal80ts KpnI
1% gel electrophoresis of plasmid
pUAST-UAS-TH
A) pUAST-UAS-TH
B) pUAST-UAS-TH BglII
Ignis Fly

2017 Tongji_China iGEM Team

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