Difference between revisions of "Team:SPSingapore/Notebook-Week-4"

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<h3 style ="margin-left:20px;">Week 4 (14/6 - 20/6)</h3>
  
  

Revision as of 13:40, 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