Difference between revisions of "Team:SPSingapore/Notebook-Week-17"

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Revision as of 12:33, 18 September 2015


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
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


▪ Sep 14

Anaerobic Promoter   ☀ Yi Han ☀

Ran gel for Chi Yan 13/9 9pm and 11pm
▶ Colony PCR no bands
▶ B1-12 no bands
▶ A1-16 bright band , correct size, extract


Anaerobic Promoter   ☀ Chi Yan ☀

PCR of invlloF1/lloendBBsuffixR with invD

Buffer 160
MgCl2 32
dNTPs 32
Primers 32/32
Template 18.3
H2O 517.7
Taq 8
Total 800

PCR of invloF2/lloendBBsuffix R with invlloF1endsuffixR
Buffer 80
MgCl2 16
dNTPs 16
Primers 16/16
Template 100
H2O 152
Taq 4
Total 400

Cycle conditions as of Chi Yan 13/9
Positions of tubes, A-C B-D
PCR done with GoTaq kit


Anaerobic Promoter   ☀ Yi Han ☀

Screen placgfp colonies for gfp using microscope
▶ Colonies 1, 4, 8 look promising

Miniprepped placgfp colonies
RE of colony 1, 4, 8
Template 1
EcoRI/PstI 0.1/0.1
Buffer 1
H2O 8
Total 10.2


Anaerobic Promoter   ☀ Chi Yan ☀

Sent colonies 1, 4, 8 of placgfppSB1C3 for sequencing with FP_BB_Prefix, RP_BB_Suffix, VF2.
Ran gel for YH placgfp pSB1C3 colonies RE 1, 4, 8
Incomplete digestion as only digested for 1.5hours
PCR of placgfp pSB1C3 colonies 1, 4, 8 from plasmids and with placfp pAC with FP_BB_Prefix and RP_BB_Suffix
Annealing temp 55degrees for 30s, extension for 60 seconds, for 25 cycles
GOTaq Master Mix 1
Buffer 20
MgCl2 10
dNTPs 4
Primers 4/4
Taq 1


Anaerobic Promoter   ☀ Yi Han ☀

PCR with GoTaq kit to make Fragment 8 from Fragment 1
Template (500ng) 40
dNTPs 4
MgCl2 16
Primers 2/2
GoTaq 0.5
PCR Buffer 20
H2O 19.5
Total 104.5


Continued from previous
Ran gradient at annealing temperature for 55-65, in wells 1, 4, 7, 12

Ran gel, no bands in all
    1. Made more F1 invsuffixendloger from invD
    2. Use BB PrefixpBirBinvstart/invendBBSuffixend to generate whole fragment
    3. Make fragment 8 using F8/invendsuffixlonger

Set PCR machine to 10 cycles at Ta gradient 40-55 degrees
30 cycles Ta 60 degrees
Ramp of 10%

PCR mix for 1 and 2
InvD Template 9.2
dNTPs 20
MgCl2 16
Primers 4/4
GoTaq 0.5
H2O 105.8

PCR mix for 3
Template 45
dNTP 8
MgCl2 8
Primers 1/1
GoTaq 1
PCR Buffer 20
H2O 16
Total 100

▶ Result: 1, 2 had clear bands, 3 had no bands


Anaerobic Promoter   ☀ Chi Yan ☀

PCR of placgfp colonies 1, 4, and 8 plasmids and placgfp pAC colony plasmid using VF2/VR
Annealing temperature is 52degrees for 30seconds, extension of 60s 30 cycles using GoTaq kit
Buffer 20
MgCl2 16
dNTPs 4
Primers 4/4
Taq 1

▶ Colony 1: Template 0.4/12.35 H2O
▶ Colony 4: Template 0.8/11.85 H2O
▶ Colony 8: Template 0.7/12.05 H2O
▶ pAC: Template 2.5/10.25