Difference between revisions of "Team:SPSingapore/Notebook-Week-15"

 
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         <li><a href = "https://2015.igem.org/Team:SPSingapore/Notebook-Week-15"><span>Week 15 (30/8 - 5/9)</span></a></li>
 
         <li><a href = "https://2015.igem.org/Team:SPSingapore/Notebook-Week-15"><span>Week 15 (30/8 - 5/9)</span></a></li>
 
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Latest revision as of 23:19, 18 September 2015


Research Notebook

Week 15 (30/8 - 5/9)

▪ Aug 30

Anaerobic Promoter    ☀ Yi Han & Chi Yan ☀

PCR with above template using lloendR/invF3
H20 140ul
Buffer 20ul
dNTP 8ul
F/R Primer 2ul/2ul
MgCl2 8ul
Template 80ul(800ng)(250ng/tube)
Taq 1ul

▶ 4 tubes. Nanodrop (1224ng/ul).
1 tube kept for running gel
3 tubes purify from suspension using Thermo(118.7 ng/ul overall). use at least 20ul elution buffer
▶ Fusion PCR with hotstartaq DNA pol Primers invlloF3 + lloEndBBSuffix
1. 200ng/tube for each template
2. 100ng/tube
3. 200ng/tube for each template + Q solution
▶ First run 10 cycles without primers. Extension for 30s
Then add primers, change settings to 95 deg C 10sec start, run 30 cycles.
Extension for 4.46. Ta of 65 deg C

Miniprep 3 tubes of invD plasmid. 218ng/ul of 150ml
1 2 3
PCR mix 200ng(1) 100ng(2) 200ng + Q solution(1)
Buffer 20ul 20ul 20ul
dNTP 8ul 8ul 8ul
MgCl2 8ul 8ul 8ul
F/R Primer 2ul/2ul 2ul/2ul 2ul/2ul
Taq 1ul 1ul 1ul
Q solution - - 40ul
lloendF1 / lloBBsuffix 3.5ul 1.75ul 3.5ul
F3 4ul 2ul 4ul
H20 151ul 155ul 151ul

Total = 200 ul/each

▶ Ran gel: smear for all
▶ After consulting Elvin (clear band of correct size should be produced) Reran above Fusion PCR for condition 2, with Ta at 58 deg C, extension time 5min
▶ Remade more Fragment 3 as with earlier reaction


▪ Aug 31

Anaerobic Promoter    ☀ Chi Yan ☀

Ran gel for fusion PCR and Fragment 3
2 large 2% gels, 120V 60min:
  (1) Fragment 3.
    100bp, 1kb with F3 contamination, F3 1kb, 100bp,
  (2) 4.4kb
    1kb, 100bp, 8 lanes & 4 kb, 1kb, 100bp
Yi Han -> Smears for F3 and 4.4kb fragment. Oh no!
  Redo PCR for F3 with less template, less primer
  previously 250ng/tube 50, 100, 150ng
  primer 0.1uM concentration, 0.1, 0.05 uM

Permutations:
0.1uM primer 50ng
0.1uM primer 100ng
0.1uM primer 150ng
0.05uM primer 50ng
0.05uM primer 100ng
0.05uM primer 150ng
Buffer 20ul
dNTP 8ul
MgCl2 8ul
Primers (0.1uM)->2/2||(0.05uM)->1/1
Template (50ng/tube|3.57ul)||
(100ng/tube|7.14ul)||
(150ng/tube|10.7ul)
Taq 1ul
H20 158/155/151 ul

▶ 28 cycle (Ta is 58, extension of 3min) did (1), (2), (4)
▶ 2 1% gels double row


Invasin + Listerolysin    ☀ Yun Ting ☀

PCR
H20 326 ul
Buffer 40ul
MgCl2 16ul
Primers 4ul/4ul
dNTP 16ul
Template 8ul
Total 400ul


PCR for placInv, 8 rxn. Primers used are FP1 placInv and lloend-R. Template is InvLLO plasmid miniprep (115ng/ul).
No bands.
H2O 310
Buffer 40
dNTPs 16
MgCl2 16
Primers 4+4
Template 8
Taq 2


Anaerobic Promoter    ☀ Yi Han ☀

Ran gel, only primer dimers. Lowering concentration of template or primer did not help -> no primer
Purified F1 (32.6ng/ul) and F2 (63.1ng/ul) from suspension
▶ F2 and F3 were PCRed out using same reaction conditions as 30/8
▶ F1/F2 template total 1000ng


ESA Quorum Sensing    ☀ Kenneth ☀

Gel extraction of the sequences:
▶ Term Tube 1: 0.237g -> 25.2ng/ul
▶ EsaR Tube 2: 0.182g -> 164.2ng/ul
▶ EsaRBS Tube 3: 0.202g -> 10ng/ul
▶ GFP Tube 4: 0.224g -> 118ng/ul
dH2O 92.56ul
Buffer 40ul
MgCL2 16ul
dNTP 16ul
FP_BBP_Term 8ul
RP_BBS_lacP 8ul
GoTaq 1ul
term (400ng) 16ul
EsaR (400ng) 2.4ul
Total 200ul
dH2O 72.82ul
Buffer 40ul
MgCL2 16ul
dNTP 16ul
FP_BBP_Term 8ul
RP_BBS_lacP 8ul
GoTaq 1ul
term (400ng) 40ul
EsaR (400ng) 3.38ul
Total 200ul


Anaerobic promoter Brown    ☀ Chi Yan ☀

Redo 29/8 to get inv F1
Tm 58degrees, 3 min extension, 35 cycles, 2ulTaw labelled CY


▪ Sep 1

Anaerobic Promoter    ☀ Yi Han ☀

Ran gel from CY 31/8 -> gel extract, PCR to get F2 with invlloF2/lloendR
dH2O 115
Buffer 20
MgCL2 8
dNTP 8
Primers 2/2
GoTaq 0.5
Template 45
Total 200
▶ CY ran the gel from above to get F2
▶ YH verified size was correct despite smear being present and gel extracted band of appropriate size
▶ PCR of F3 from F2 using invlloF3 and BBprefix-invasin Suffix
dH2O 125
Buffer 20
MgCL2 8
dNTP 8
Primers 2/2
GoTaq 0.5
Template 35
Total 200


Invasin + Listerolysin    ☀ Yun Ting ☀

Reran PCR from 31/8 for placInv, 8 rxn. Used thermogradient for annealing temperature – higher Ta near 65deg seems to work better.
▶ Ran gel with 20ul per well – bands just below 3kb.
▶ Cut out 7 bands, placed in 2 gel extraction columns. Eluted with H2O that has been warmed to 55deg. Poor yield– 15ng/ul.

Reran PCR from 31/8 for placInv, 4 rxn. Adjusted thermogradient for Ta= 60, 62, 65, 67deg for 40cycles O/N. Generally faint bands, especially at 60deg.
dH2O 169
Buffer 20
MgCL2 8
dNTP 8
Primers 2/2
GoTaq 1
Template 2
Total 200


▪ Sep 2

Anaerobic Promoter    ☀ Chi Yan ☀

Ran gel from YH 1/9 F3 and BBPrefix-inv-BBsuffix
    1% 100v 45min
    1kb 100bp F3 BBPre-inv-BBsuffix 100bp 1kb

Result: Smear
Ran gel for Cy 31/8 1% 100v 45min
Gel extract


Anaerobic Promoter    ☀ Yi Han ☀

PCR to get F1 as of 29/8
Pcr to get F2 as with 1/9 using gradient PCR
Ran gel @ 4pm, Ran PCR to get F3, inv fragment


Invasin + Listerolysin    ☀ Yan Ting ☀

Gel extraction
nanodrop results 1: 9.8ng/ul 2: 3.0ng/ul


Anaerobic Promoter    ☀ Yi Han ☀

PCR of placgfp with BBPrefixplacGFP-F/BBSuffixGFP-R
dH2O 100
Buffer 20
MgCL2 8
dNTP 8
Primers 2/2
GoTaq 1
Template 60
Total 200

Repeat PCR for inv fragment as of 1/9
Ran gel for above samples
▶ No PCR product for placgfp
▶ Smear for invasin fragment
▶ Band of correct size for F3 - gel extract
▶ Amplify more F3 from F3
PCR Reaction
▶ Template-> gel extracted F3
▶ Primers invlloF3/lloendR
dH2O 298
Buffer 40
MgCL2 16
dNTP 16
Primers 4/4
GoTaq 2
Template 20
Total 400


Invasin + Listerolysin    ☀ Yun Ting ☀

PCR 1.4 for placInv, 10 rxn. Primers used are FP1 placInv and lloend-R. Template is InvLLO plasmid miniprep (115ng/ul).
Ta= 58-64deg, Extension time=3.5min.


▪ Sep 3

Anaerobic Promoter    ☀ Chi Yan ☀

Miniprep of placgfp then gradient PCR for placgfp following YH 2/9 with BBPRefixplacgfp-F and BBSuffixGFP-R
dH2O 288
Buffer 40
MgCL2 16
dNTP 16
Primers 4/4
GoTaq 2
Template 35
Total 400

Gradient 58-63 degrees
Chose: 58, 58.4, 58.8, 60, 60.7, 61.3, 62.4, 62.9


Anaerobic Promoter    ☀ Yi Han ☀

Ran gel for F3 from F3 => Smear
PCR for fusion fragment
  1:1 ratio of template
  500ng Front fragment (5ul)
  500ng back fragment (5ul)
Buffer20
MgCL28
dNTP8
Primers2/2
Taq1
H2O44
Total100


Anaerobic Promoter    ☀ Chi Yan ☀

Yun Ting's PCR Nanodrop
    1. 811
    2. 722
    3. 730
    4. 710
    5. 727
Ran gel for Yun Ting’s PCR (5/7 and cut bands), Yi Han's 3/9 fusion pcr (no bands) 100v 40 min


ESA Quorum Sensing    ☀ Kenneth ☀

PCR purification of 31/8


Anaerobic Promoter    ☀ Chi Yan ☀

PCR to get F2 and F3
invlloF2/lloendR
dH2O123
Buffer20
MgCL28
dNTP8
Primers2/2
GoTaq1
Template36
Total200
invlloF3/lloendR
dH2O306
Buffer40
MgCL216
dNTP16
Primers4/4
GoTaq2
Template12
Total400

58 degrees annealing, 42 cycles, 3min extension

PCR of placgfp with BBPrefixplacGFP-F/BBsuffix GFP-R and GFP-R/BBPrefixplacgfp-F as control (25ul)

Half of normal MgCl2 concentration used, rediluted all 3 primers Gradient PCR 55 to 65 degrees
BBPrefixplacGFP-F/BBsuffix GFP-R
dH2O218.2
Buffer30
MgCL26
dNTP12
Primers3/3
GoTaq1.5
Template26.3
Total200

GFP-R/BBPrefixplacgfp-F
dH2O18.2
Buffer2.5
MgCL22.5
dNTP1
Primers0.25/0.25
GoTaq0.13
Template2.2
Total25

Inoculated 4 tubes 3mL LB + ch1 placgfp


Anaerobic Promoter    ☀ Yi Han ☀

Ran gel for F2 (product), F3 (smear) and placgfp (no product)
  Smear for F3 faint band for F2
  gel extract F2
PCR using GoTaq kit for F3 and inv fragment
dH2O53.5
Buffer10
MgCL24
dNTP4
Primers4/4
GoTaq0.5
Template10
Total100

PCR with hotstart and inv
5 cycles with Ta 46 first
30 cycles overnight at Ta 60
dH2O27.5
Buffer4
MgCL24
dNTP1
Primers1/1
GoTaq0.5
Template2
Total50


Remake F2
dH2O144
Buffer20
MgCL24
dNTP4
Primers2/2
GoTaq1
Template15
Total200
Remake F1
dH2O333
Buffer40
MgCL28
dNTP8
Primers4/4
GoTaq2
Template9
Total400
▪ Sep 4

Invasin + Listerolysin    ☀ Yun Ting ☀

PCR prefix-placinv for 10 reactions
dH2O 348
Buffer 40
MgCL2 16
dNTP 16
Primers 4/4
GoTaq 2
Template 10
Total 400

Ran gel at 110V, 1h. Marker was not distinct. No bands.

PCR 2.1 for prefix-plac-Inv-suffix, 8 rxn. Primers used are FP2_prefixplac and RP_Invendsuffix. For tubes 1-4, template is PCR product 1.4.10 (i.e. tube 10 from PCR reaction 1.4). For tubes 5-8, template is gel extracted PCR product 1.2 (i.e. gel extract from PCR reaction 1.2). Ta= 59, 61, 62, 64deg.
No bands.

H2O 310
Buffer 40
dNTPs 16
MgCl2 16
Primers 4+4
Template 8
Taq 2


PCR 2.2 for prefix-plac-Inv-suffix, 4 rxn. Primers used are FP2_prefixplac and RP_Invendsuffix.
Template is PCR product 1.4.10 (i.e. tube 10 from PCR reaction 1.4).
Thermocycler 5 cycles at Ta=27deg (Ta of FP2), then 30 cycles at Ta= 58 & 60deg.
No bands.


▪ Sep 5

Anaerobic Promoter    ☀ Yi Han ☀

Ran gel for inv fragment, F1, F2
  Smear for inv fragment, product for Fa and F2 gel extract
  Trying again to make inv fragment
  5 cycles at 46 degrees, 30 cycles at 59 degrees
Inv fragment
dH2O3.75
Buffer5
MgCL22
dNTP2
Primers1/1
GoTaq0.25
Template15
Total50

PCR for placgfp
  5 cycles at Ta 40 degrees
  30 cycles at Ta 30 defrees and 60 degrees (12 tubes from 200ul of PCR reaction mix)
dH2O124
Buffer20
MgCL28
dNTP8
Primers4/4
GoTaq1
Template31
Total200


PCR for F2
dH2O125
Buffer20
MgCL28
dNTP8
Primers4/4
GoTaq1
Template20
Total200


Invasin + Listerolysin    ☀ Yun Ting ☀

PCR 2.2 PrefiplacInv for 2 reactions
Addition of 5 cycles at Ta=27 degrees before 30cycles with (Ta 58 & 60 degrees)


Anaerobic Promoter    ☀ Yi Han ☀

Results of PCR -> Faint band of F2-> gel extract
  no product still smear for inv fragment
  Clear band for placgfp-> gel extract (no amplification)
PCR to make inv fragment from F3 and PCR for more F2
  5 cycles at 30, 5 cycles at 40, 30cycles at 55 degrees Ta
Invasin fragment
dH2O105
Buffer20
MgCL28
dNTP8
Primers4/4
GoTaq1
Template50
Total200

▶ Ran gel, result is no amplification

RE of linearised backbone of pSB1C3 and placgfp fragment with BB prefix and Suffix
125 ng linearised backbone (2kb) 5
10X buffer 2
EcoRI 0.2
PstI 0.2
H2O 12.6
Total 20

250 ng pcr product 7.6
10X buffer 2
EcoRI 0.2
PstI 0.2
H2O 10
Total 20


Ran RE for 3 hours
PCR to make more F1
dH2O 293
Buffer 40
MgCl2 8
dNTP 8
Primers 4/4
GoTaq 1
Template 17
Total 400


ESA Quorum Sensing    ☀ Adrian ☀

RE digest:
EsaR thing 29.1
10X buffer 4.5
EcoRI 1
SpeI 1
BSA 4.5
H2O 4.9
Total 45
GFP Thing 13.3
10X buffer 2
EcoRI 1
PstI 1
H2O 2.7
Total 20


Ligation
Vector 2
Insert 1 4
Insert 2 5.58
10X buffer 2
Ligase 1
H2O 5.42
Total 20

▶ 5ul of ligated product was transformed into dH5 alpha