Team:SPSingapore/Notebook-Week-17


Research Notebook

Week 12 (13/9 - 18/9)

▪ Sep 13

Invasin + Listerolysin   Anaerobic Promoter
☀ Chi Yan ☀

Ran gel for placgfp-psB1C3 colonies from Chi Yan 12/9 11pm. 1% 110V 30min
▶ no bands
▶ no positive colonies


Ran gel for PCRs to make Prefix-pNirB-inv-Suffix from Chi Yan 12/9 11pm
▶ E: successful, cut bands.
▶ F, G, H,I no bands


PCR for YT, Template used is gel extract ‘1.5.2’ (26ng/ul)
Primers used are FP2’ and RP2


8 cycles 50-60degrees 2 min for annealing step
30 cycles 55-65 degrees 40s for annealing step


50ul, 3 tubes, put in columns 4, 8 12. Negative control added.
Buffer 15
dNTPs 6
MgCl2 6
Primers 1.5/1.5
Template 15
H2O 104.25
Taq 0.75


Invasin + Listerolysin   Anaerobic Promoter
☀ Yi Han ☀

Ran gel for YT PCR

PCR using F2 to produce F2/invendsuffix
Primer pairs are invlloF3new/invendsuffix and invlloF6/invendsuffix


PCR for YT, Template used is gel extract ‘1.5.2’ (26ng/ul)
Primers used are FP2’ and RP2

invlloF3new/invendsuffix
Template (1000ng) 18
Primer 2/2
Buffer 20
sNTP 8
Taq 1
H2O 149
Total 200

invlloF6/invendsuffix
Template 500ng 9
Primer 1/1
Buffer 10
dNTP 4
Taq 0.5
H2O 74.5
Total 100


Attempt to make more of inv fragment from Yun Ting’s gel extract
Primers are FP2’/RP
Template 8.2
Primer 1/1
Buffer 10
dNTP 4
Taw 0.5
H2O 75.3
Total 100


Invasin + Listerolysin   Anaerobic Promoter
☀ Chi Yan & Yi Han ☀

PCR of invlloF1/lloendBBSuffix R with invasin D plasmid as template
▶ 5 cycles gradient 40-50degrees
▶ 20 cycles 62 degrees


Buffer 40
MgCl2 16
dNTPs 16
Primers 4/4
Template 9.2
H2O 308.8
Taq 2
Total 400

▶ 12 tubes at each temp, 33ul each

For YT: PCR of RP2/FP2’ using gel extract 1.6

Buffer 20
dNTP 8
MgCl2 8
Primers 2/2
Taq 1
H2O 135
Total 200
(3 tubes + 5ul template, 1 tube control)


Gel extract F1 invlloendsuffix
PCR of invlloend/BBsuffixR with invD
PCR of invlloF2/lloendBBsuffixR with F1/invlloendsuffix


F1invlloendsuffix 20
BUffer 80
MgCl2 32
dNTPs 32
Primers 8/8
Template 18.4
H2O 617.6
Taq 4
Total 800

Buffer 20
MgCl2 8
dNTPs 8
Primers 2/2
Template 84
H2O 75
Taq 1
Total 200

▶ 5 cycles 44-48 degrees
  25 cycles 60degrees


Colony PCR new placgfp-pSB1C3 following CY 12/9 11pm
Primers FP_KpnI_GFP/GFP_BBSuffix_new
60 degrees 25 cycles