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&#9642; June 20
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<span class = "blue"> Invasin + Listerolysin</span>
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Freezing of HEK293
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<br> P6
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Revision as of 19:31, 17 September 2015


Research Notebook

Week 4 (14/6 - 20/6)

▪ June 14

Anaerobic Promoter    ☀ Yi Han ☀

PCR
▶ RP_XhoI_GFP and FP_KpnI_GFP with GFP-3 as template
▶ for 7 reactions with control
Mastermix * 8
PCR buffer 80ul
Primers 0.8ul/0.8ul
DNA polymerase 4ul
dH2O 494ul
Templates 38.75


Digest more plasmid (Esa plasmid)
4 rxns ( KpnI/XhoI digest) 50ul each
Buffer 20ul
KpnI 2ul
XhoI 2ul
DNA 34.8ul
H20 141.2ul


▪ June 15

Anaerobic Promoter    ☀ Yun Ting ☀

PCR: RP_XhoI_GFP and FP_KpnI_GFP with GFP midiprep for 15 reactions with control:
PCR buffer 170ul
Primers 1.7ul, 1.7ul
dNTP 34 ul
DNA polymerase 8.5 ul
dH2O 1127.1ul
Templates 17ul
Total 1360ul

PCR protocol “GFP 1” (32 cycles)


Anaerobic Promoter    ☀ Yi Han ☀

GFP PCR product -> 448ng/ul
Gel extraction
Qiagen gel extraction kit at MBI
Esa gel band from 14/6
Nanodrop: 16.4 ng/ul, 260/280 = 2.60

Plasmid construction
RE digest (KpnI, XhoI) 3 Replicates of the following:
DNA 2.6ul
Buffer 5ul
H2O 41.5ul
KpnI 0.5ul
XhoI 0.5ul


Ligation (2x reaction) 40ul
10X T4 DNA ligase buffer 4ul
Vector DNA (16ng/ul) 100ng -> 6.25ul
insert DNA (75ng) 12 ul
T4 DNA Ligase 2ul
H2O 16ul


▶ Note: ALL digested DNA in tube labeled lacGFP.ligation was used
▶ Nanodrop of ligation: 1114.0ng/ul. 260/280 = 3.7


▪ June 16

Anaerobic Promoter    ☀ Yi Han ☀

Transformation of ligated esa-GFP plasmid into DH5\alpha cells
DH5alpha, unlabelled brown vial inside DH5\alpha box at -80
▶ Plates spread at 1140h
▶ LB + chloroamphenicol
     4x (100ul)
     10x (40ul)
     20x (20ul)
     LB - 20x (20ul)
Remaining transformed cells (~220ul) are kept at 4C
▶ labeled as placGFP in brown tube


▪ June 17

Anaerobic Promoter    ☀ Yi Han ☀

only 4x placGFP plate has colonies (7)
▶ spread one new LB + chlor plate with 200ul of transformed bacteria
▶ picked 6 colonies to grow for miniprep in LB + chl liq media
▶ Conclusion: Protocol works but optimization for increase vol needed
Cloning of synparts in amp vector
▶ comes as 4mg dry DNA
▶ +40ul of DI/RNAse free H20
▶ for transformation, 150ml wed

Miniprep of placGFP followed by RE digest (Kpn, XhoI): 50 ul total
Buffer 2.5
KpnI 1
XhoI 1
DNA 10
H20 120.25


Colony PCR for synparts
▶ Failed
▶ No specific bands produced
▶ Might need optimization


Invasin + Listerolysin    ☀ Yan Ting ☀

Subculture of HEK 293
▶ P4 -> p5
▶ grow/split into 150mm dish for freezing on sat (20/6)
     30ml DMEM + 1ml cells
▶ 90mm dish for maintenance (buffer)
     10ml DMEM + 30ul cells
▶ Cells combined from 3 90mm dishes


▪ June 19

esa Quorum Sensing    ☀ Yi Han ☀

RE digest (XhoI, KpnI) of esa and GFP


esa Quorum Sensing    ☀ Yun Ting ☀

Gel electrophoresis (100V, 40min).
▶ Lane 2: gpf: no bands
▶ Lane 3: 100bp ladder
▶ Lane 4: blank
▶ Lane 5: esa: 2 bands
▶ Lane 6: 1kb ladder
▶ Lane 7:blank


▪ June 20

Invasin + Listerolysin    ☀ Yan Ting ☀

Freezing of HEK293
P6
cells from a 150 mm dish and 90mm dish