Revision as of 18:07, 18 November 2015

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Lab Note

Yeast With IL-8 Receptor

2015.7.1

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Extraction of gDNA of Far1∆::KANMX strain
  2. Detection the concentration of fast extracted gDNA of ∆Far1 strain
  3. PCR gDNA of Far1∆ ::KANMX
  4. Electrophoresis to check PCR product

Experiment steps:
< Extraction of gDNA of Far1∆::KANMX strain>
Consult the protocol


Strains	260/280	260/230	C(ng/μl)
Far1∆::KANMX strain	1.62	0.97	44.7

1. Design of primers


Name	Pur.	Seq.(5’-3’)	Size (mer.)	MW (g/mol)	Tm (℃)	GC (%)	Nmole	μl for 100μM
dFAR1 F’	Desalt	ggTTTTgTTAggCgggCAAg	20	6244.1	53.8	55	21.25	212.50
dFAR1 R’	Desalt	CATTAACTgCTATTTACgACgC	22	6669.4	51.1	41	17.12	171.20

2. PCR program


Step	Temperature	Time
Step1	95℃	5min
Step2	95℃	30s
Step3	52℃	30s
Step4→step2 for 30 cycle	72℃	2min
Step5	72℃	5min
Step6 (hold on)	10℃	1hr

3.PCR reagent


10x Dream Taq buffer	2.5μl
2.5mM dNTP	0.5μl
10mM primer(F)	0.5μl
10mM primer(R)	0.5μl
template (Far1∆::KANMX strain gDNA)	3.4μl
Taq polymerase	0.5μl
ddH2O	17.1μl
Total volume	25μl

< Electrophoresis to check PCR product>
Material:
  DNA marker: 100bp ladder 8μl
  DNA sample: 2μlPCR product + 1μl 6x loading buffer
Condition:
  0.5xTBE buffer 100V
Time:
  30min
Result:
  M：Marker；#1：Far1 ∆ for annealing at 52℃
  There is no band appears in the gel electrophoresis.
Conclusion:
  Due to the Figure 1 in result, we have to check the temperature of primers annealing and the design of primers to solve the problem. Next, we use gradient PCR to find out the exactly temperature of primer annealing and we add positive control as well.

2015.7.2

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Extraction of gDNA of FAR1∆::KANMX strain
  2. Detection the concentration of fast extracted gDNA of FAR1∆::KANMX strain
  3. First round of PCR
  4. Electrophoresis to check first round-PCR product
  5. Second round of PCR

Experiment steps:
< Extraction of gDNA of FAR1∆::KANMX strain>
Consult the protocol


Strains	260/280	260/230	C(ng/μl)
Far1∆::KANMX	1.72	0.78	42.7
Positive control	1.63	0.76	37.1

1. Information of primers


Name	Pur.	Seq.(5’-3’)	Size (mer.)	MW (g/mol)	Tm (℃)	GC (%)	Nmole	μl for 100μM
dFAR1 F’	Desalt	ggTTTTgTTAggCgggCAAg	20	6244.1	53.8	55	21.25	212.50
dFAR1 R’	Desalt	CATTAACTgCTATTTACgACgC	22	6669.4	51.1	41	17.12	171.20

2.PCR program


Step	Temperature	Time
Step1	95℃	5min
Step2	95℃	30s
Step3	Gradient42℃-46℃-50℃	30s
Step4→step2 for 30 cycle	72℃	2min
Step5	72℃	5min
Step6 (hold on)	10℃	1hr

3.PCR reagent


10x Dream Taq buffer	2.5μl
2.5mM dNTP	0.5μl
10mM primer(F)	0.5μl
10mM primer(R)	0.5μl
template (Far1∆::KANMX strain gDNA)	3.4μl
Taq polymerase	0.5μl
ddH2O	17.1μl
Total volume	25μl

< Electrophoresis to check first round-PCR product (25μl ul)>
Material:
  DNA marker: 100bp ladder 8μl
  DNA sample: 2μl PCR product + 1μl 6x loading buffer
Condition:
  0.5xTBE buffer 100V
Time:
  30min
Result:
M: Marker; #1: FAR1∆ annealing at 42 ℃; #2: FAR1∆ annealing at 46 ℃;
#3: FAR1∆ annealing at 50 ℃; #4: FAR1∆ annealing at 52 ℃(positive control)

1.PCR program


Step	Temperature	Time
Step1	95℃	5min
Step2	95℃	30s
Step3	46℃	30s
Step4→step2 for 30 cycle	72℃	2min
Step5	72℃	5min
Step6 (hold on)	10℃	1hr

2.PCR reagent


10x Dream Taq buffer	5μl
2.5mM dNTP	1μl
10mM primer(F)	1μl
10mM primer(R)	1μl
template (First round PCR product)	1μl
Taq polymerase	1μl
ddH2O	40μl
Total volume	50μl

2015.7.3

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. 1.Electrophoresis to check second round-PCR product
  2. Transform PCR product into FUS::GFP(His) strain
Experiment steps:
< Electrophoresis to check second round-PCR product (50μl)>
Material:
  DNA marker: 100bp ladder 8μl   DNA sample:2μl PCR product + 1ul 6x loading buffer Condition: 0.5xTBE buffer 100V
Time: 30min

Result:
Figure 1. Gel electrophoresis of FAR1∆
M: Marker; #1: FAR1∆ annealing at 46 ℃

Consult the protocol < protocol of yeast transformation>
Use strain name: FUS1-GFP(His)
Selection plate: YPD+G418

2015.7.6

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Check transformation result
  2. Second round PCR
  3. Electrophoresis to check PCR product
  4. Incubate E.coli with shuttle vector-p426GAL1 from stock
Experiment steps:

1. Take plates out from incubator and observe growth of colony
2. Conclusion: We failed to transformation of PCR product so we should it again.

Consult the experiment record <2015.7.2 Experiment Record>

< Electrophoresis to check second round-PCR product>
Material:
  DNA marker: 100bp ladder 8μl
  DNA sample:2μlPCR product + 1μl 6x loading buffer
Condition: 0.5xTBE buffer 100V
Time: 30min
Result:

Conclusion: Its expected length is 1.9kb and it worked.

< Incubate E.coli with shuttle vector-p426GAL1 from stock>
1. Take stock E.coli with shuttle vector-p426GAL1 from -80℃ fridge.
2. Plate E.coli on LB+Amp plates.
3. Incubate in 37℃ overnight.

2015.7.7

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
1. Transform PCR product into FUS::GFP (His) strain
Experiment steps:

Consult the experiment record <2015.7.3 Experiment Record>

2015.7.9

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
1. Miniprep plasmid of p426GAL1
2. Measure concentration of plasmid.

Experiment steps:
< Miniprep plasmid of p426GAL1
Consult the protocol


	concentration	260/280	260/230
P426GAL1/1	130.9ng/μl	1.91	2.36
P426GAL1/2	121.3ng/μl	1.89	2.26

2015.7.12

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
1. Second round PCR
2. Incubate FAR1△::KANMX strain in 5ml YPD+A medium.
Experiment steps:

Consult the experiment record <2015.7.2 Experiment Record>

2015.7.17

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
1. Transform PCR product into FUS1-GFP strain
Experiment steps:
< Transform PCR product into FUS1-GFP strain>
Consult the experiment record <2015.7.3 Experiment Record>

2015.7.20

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Maintain the colonies of far1Δ::KanMX-FUS1-GFP.
  2. 2nd round PCR for far1Δ::KanMX
  3. Phenol chloroform and EtOH precipitation of 2nd round PCR product
Experiment steps:
< Maintain the colonies of far1Δ::KanMX-FUS1-GFP >
  1. Choose 10 colonies to transfer the new YPD+G418 plate.
  2. Check plates after two days.

<2nd round PCR for far1Δ::KanMX
1.PCR program


Step	Temperature	Time
Step1	95℃	5min
Step2	95℃	30s
Step3	46℃	30s
Step4→step2 for 30 cycle	72℃	2min
Step5	72℃	5min
Step6 (hold on)	10℃	1hr

2.PCR reagent


10x Dream Taq buffer	5μl
2.5mM dNTP	1μl
10mM primer(F)	1μl
10mM primer(R)	1μl
template (First round PCR product)	1μl
Taq polymerase	1μl
ddH2O	40μl
Total volume	50μl

< Phenol chloroform and EtOH precipitation of 2nd round PCR product >
Consult the protocol

2015.7.21

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Transform PCR product(Far1Δ::KanMX) into FUS1-GFP strain
  2. Extraction of gDNA of yeast
  3. Check PCR for far1Δ::KanMX-FUS1-GFP
  4. Digestion of p426 Gal
Experimental steps: < Transform PCR product into FUS1-GFP strain> Consult the experiment record <2015.7.3 Experiment Record> Because last time , negative control also had grown colony so we did transformation again. At the same time , we selected colony on selection plate to do PCR check.

< Extraction of gDNA of yeast>
Consult the protocol < protocol of fast extraction of gDNA of yeast>

< Check PCR for Far1Δ::KanMX- FUS-GFP >

1.PCR program


Step	Temperature	Time
Step1	95℃	5min
Step2	95℃	30s
Step3	46℃	30s
Step4→step2 for 30 cycle	72℃	90sec
Step5	72℃	5min
Step6 (hold on)	10℃	1hr

2.PCR reagent


10x Dream Taq buffer	2.5μl
2.5mM dNTP	0.5μl
10mM primer(F)	0.5μl
10mM primer(R)	0.5μl
template (First round PCR product)	8μl
Taq polymerase	0.5μl
ddH2O	12.5μl
Total volume	25μl


far1Δ::KanMX-FUS-GFP	260/280	260/230	ng/μl
1	1.76	0.91	111.7
2	2.05	1.32	158.2
3	2.02	1.29	120.4
4	1.98	1.08	89.9
5	2.05	0.95	66.9
6	2.07	1.45	182.6
7	1.86	0.90	95.4
8	2.08	1.45	166.4
9	2.18	0.42	22.5
10	2.07	1.30	102.4

< Digestion of p426 Gal >


	Eco RI(μl)	Bam HI(μl)	Uncut(μl)
ddH2O	20.5	20.5	21.5
10x NEB buf. #4	2.5	2.5	2.5
DNA(200ng)	1.5	1.5	1.5
Enzyme	0.5	0.5	--
total	25	25	25

1. Incubate at 37 for 1 hr.
2. Run the sample on the 1% agarose gel at 100 V for 30 min.(10 μl sample +2 μl loading dye)

2015.7.23

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Digestion of p426 Gal and rho-CXCR1
  2. Gel extraction of p426 Gal(vector)
  3. Clean up of rho-CXCR1(insert)
Experimental steps:
< Digestion of p426 Gal and rho-CXCR1>


	121 ng/μl	131 ng/μl
p426 Gal (2 μg)	16.5μl	15.2μl
10x NEB buf. 4	5μl	5μl
Bam HI	2μl	2μl
Eco RI	2μl	2μl
ddH2O	24.5μl	25.8μl
Total	50μl	50 μl


Rho-CXCR1	8(400 ng)
10x NEB buf. 4	5μl
Bam HI	1μl
Eco RI	1μl
ddH2O	35μl
Total	50μl

< Gel extraction of p426 Gal >
Consult the protocol < protocol of gel extraction>

< Clean up of rho-CXCR1>
Consult the protocol

2015.7.24

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
1. Ligation of the rho-CXCR1 and p426 Gal
2. Transform the ligation product into the E.coli
Experimental steps:
< Ligation of the rho-CXCR1 and p426 Gal>


	1:3	Vector only
Vector(11.1 ng/μl)	10μl	10μl
Insert(9.1 ng/μl)	7μl	0μl
Ligase	1μl	1μl
Ligation buff.	2μl	2μl
ddH2O	0μl	7μl
total	20μl	20μl

Incubate the ligation product at R.T. for 2 hr.

< Transform the ligation product into the competent cell>
Consult the protocol < protocol of ligation and transformation of E.coli>

2015.7.27

Operator: Wan-Yun, Jin-Ting
Goal:
1. Fast extraction of gDNA of Far1Δ::KanMX-FUS1-GFP
2. Check PCR for far1Δ::KanMX-FUS-GFP
Experimental steps: < Fast extraction of gDNA of far1Δ::KanMX-FUS-GFP > Consult the protocol


Strains	260/280	260/230	C(ng/μl)
FAR1∆::KANMX	1.85	1.19	177.3
FAR1∆::KANMX	1.83	1.19	163.8
Positive control	1.96	1.61	235.3

< Check PCR for Far1Δ::KanMX-FUS1-GFP>
1.PCR program


Step	Temperature	Time
Step1	95℃	5min
Step2	95℃	30s
Step3	46℃	30s
Step4→step2 for 30 cycle	72℃	90sec
Step5	72℃	5min
Step6 (hold on)	10℃	1hr

2.PCR reagent


	FAR1∆::KANMX	Positive control
10x Dream Taq buffer	2.5μl	2.5μl
2.5mM dNTP	0.5μl	0.5μl
10mM primer(F)	0.5μl	0.5μl
10mM primer(R)	0.5μl	0.5μl
template (First round PCR product)	0.92μl	0.64μl
Taq polymerase	0.5μl	0.5μl
ddH2O	19.58μl	19.86μl
Total volume	25μl	25μl

2015.7.28

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1.Fast extraction of pGAL426 rho-CXCR1
Experimental steps:
< Fast extraction of plasmid DNA >
  1. Add 50 μl of STE buffer(100 mM NaCl, 10 mM Tris buffer, pH 7.0, 1 mM EDTA) into each new Eppendorf tube.
  2. Add 50 μl of Phenol chloroform and vortex vigorously for 30 sec.
  3. Centrifuge at 13,000 g for 5 min.
  4. Remove the 10 μl of supernatant to a new Eppendorf tube and run the sample on the 1 % agarose gel.

2015.7.29

Operator: Wan-Yun, Jin-Ting
Goal:
1. Miniprep of p426 Gal-rho-CXCR1
2. Enzyme digestion to check p426 Gal-rho-CXCR1
Experimental steps:
< Miniprep of p426 Gal-rho-CXCR1>
Consult the protocol

< Enzyme digestion for the p426 Gal-rho-CXCR1 check >
1. Enzyme digestion


p426 Gal-rho-CXCR1	#7,9	+,#8,12,16
10x NEB buff.4	2.5μl	2.5μl
Eco RI	0.5μl	0.5μl
Bam HI	0.5μl	0.5μl
DNA	2μl	1μl
ddH2O	19.5μl	20.5μl
total	25μl	25μl

2. Loading 10 μl sample and 2 μl DNA loading dye into the 1 % agarose gel.

2015.7.30

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
1. Transform pSB1C3 BBa_J04450 into the competent cell
2. Transform the plasmid (pRS405) into the competent cell
Experimental steps:
< Transform pSB1C3 BBa_J04450 into the competent cell>
Consult the protocol
Selection plate: LB+ Cam plate

< Transform the plasmids (pRS405) into the competent cell>
Selection plate: LB+ Amp plate

2015.8.3

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Miniprep of pRS405 and run the 1% agarose gel
  2. Digestion of pRS405 with Xho I, Bam HI and Eco RI
  3. Digestion of p426 Gal with Xho I
  4. Inoculate the bacteria with BBa_J04450 into the LB+ chloramphanicle broth

Experiment steps:
< Miniprep of pRS405 and run the 1% agarose gel >
Consult the protocol < protocol of miniprep plamid>

< Digestion of pRS405 with Xho I, Bam HI and Eco RI >


	uncut	Xho I	Bam HI	Eco RI
pRS405 (V105○1)	0.5	0.5	0.5	0.5
10x NEB buff.4	2.5	2.5	2.5	2.5
Enzyme	-	0.5	0.5	0.5
ddH2O	22.5	21.5	21.5	21.5
total	25 μl	25 μl	25 μl	25 μl

Reaction condition: 37℃, 1hr

< Digestion of p426 Gal with Xho I >


.	uncut	Xho I
p426 Gal (131 ng/μl)	0.5	0.5
10x NEB buff.4	2.5	2.5
Enzyme	-	0.5
ddH2O	22.5	21.5
total	25 μl	25 μl

Reaction condition: 37℃, 1hr

2015.8.4

Operator: Wan-Yun, Jin-Ting
Goal:
  1. 1st PCR of rho-CXCR1
  2. Electrophoresis to check the PCR product
  3. Miniprep of pSB1C3 BBa_J04450
Experiment steps:
<1st PCR of rho-CXCR1>
1. PCR program


Step	Temp.	Time
Step1	95℃	5 min
Step2	95℃	30 sec
Step3	40,44,48℃	30 sec
Step4→Step2 for 30 cycle	72℃	80sec
Step5	72℃	5 min
Step6(Hold on)	10℃	1 hr

2. PCR reagent


10x Dream Taq buffer	2.5μl
2.5mMdNTP	0.5μl
10μMprimer(F’)	0.5μl
10μMprimer(R’)	0.5 μl
Rho-CXCR1	3μl
Taq polymerase	0.5 μl
ddH2O	17.5μl
Total volume	25 μl

< Electrophoresis to check the PCR product>
< Electrophoresis to check second round-PCR product>
Material:
  DNA marker: 100bp ladder 8μl
  DNA sample:2μlPCR product + 1μl 6x loading buffer
Condition:
  0.5xTBE buffer 100V
Time:
  30min
Result:

Conclusion: It expected length is 1200bp and it worked.
< Miniprep of pSB1C3 BBa_J04450>
Consult the protocol

2015.8.10

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Electrophoresis to check p426Gal-rho-CXCR1 digestion product again
  2. Do digestion of Lucy-rho-CXCR1 (insert) and p426Gal (vector)
  3. Clean up digestion product of Lucy-rho-CXCR1
  4. Gel extraction of digestion product of p426Gal
  5. Ligation insert and vector
  6. Transform the construct into competent cell
Experiment steps:
< Electrophoresis to check p426 Gal-rho-CXCR1 digestion product again>
Material:
  DNA marker: 1kb ladder 6ul
  DNA sample:2ul p426Gal-rho-CXCR1 digestion product(BamHI and XhoI) + 1ul 6x loading buffer
Condition:
   0.5xTBE buffer 100V
Time:
  30min Result:

Note: pGAL426 rho-CXCR1 enzyme digestion product with BamHI and XhoI


	Lucy-rho-CXCR1 (insert)	p426Gal (vector)	p426Gal (vector)
10x NEB buffer#4	5μl	5μl	5μl
BamHI	1μl	1μl	1μl
XhoI	1μl	1μl	1μl
DNA	25μl (400ng)	7μl (2ug)	7μl
ddH2O	18μl	34μl	34μl
Total volume	50μl	50μl	50μl

Reaction condition : 37℃ for 1hr
< Clean up digestion product of Lucy-rho-CXCR1>
Consult the protocol


Conc	260/280	260/230
10.0ng/ul	1.95	0.94

< Gel extraction of digestion product of p426Gal>
Consult the protocol


Conc.	260/280	260/230
11.2ng/ul	1.75	0.20

< Ligation insert and vector>
Vector:insert=1:3


	Test tube	Vector only
Vector(11.2ng/ul)	9μl	9μl
Insert(10.0ng/ul)	6μl	0μl
Ligase	1μl	1μl
10xLigation buffer	2μl	2μl
ddH2O	2μl	8μl
Total volume	20μl	20μl

Reaction condition: Room temperature for 2-4hr

< Transform the construct into competent cell>
Selection plate: LB+Cam

2015.8.11

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Obeserve colony growth to confirm transformation result
  2. PCR Gpa1-Leu2
  3. Electrophoresis to check PCR product
  4. Second PCR round of Gpa1-Leu2 with first product
  5. Extraction of DNA with Phenol/Chloroform
Experiment steps:
< Obeserve colony growth to confirm transformation result>
Select 12 colonies and incubate in LB broth(with Amp) overnight,37℃

< PCR Gpa1-Leu2>
1.PCR program


Step	Temperature	Time
Step1	95℃	5min
Step2	95℃	30s
Step3	48℃,53℃,59℃,62℃	90s
Step4→step2 for 30 cycle	72℃	5min
Step5	72℃	5min
Step6 (hold on)	10℃	1hr

2.PCR reagent


10x Dream Taq buffer	2.5μl
2.5mM dNTP	0.5μl
10mM primer(F)	0.5μl
10mM primer(R)	0.5μl
Template(pRS405,150ng)	0.55μl
Taq polymerase	0.5μl
ddH2O	20μl
Total volume	25μl

< Electrophoresis to check second round-PCR product (50μl)>
Material:
  DNA marker: 1kb ladder 6μl
  DNA sample:2μl PCR product + 1μl 6x loading buffer
Condition:
  0.5xTBE buffer 100V
Time:
  30min
Result:

Conclution: Its expected length is 1502bp and it worked.

< Second PCR round of Gpa1-Leu2 with first product>


Step	Temperature	Time
Step1	95℃	5min
Step2	95℃	30s
Step3	53℃	30s
Step4→step2 for 30 cycle	72℃	5min
Step5	72℃	5min
Step6 (hold on)	10℃	1hr

2. PCR reagent


10x Dream Taq buffer	5μl
2.5mM dNTP	1μl
10mM primer(F)	1μl
10mM primer(R)	1μl
template (First round PCR product)	1μl
Taq polymerase	1μl
ddH2O	40μl
Total volume	50μl

< Extraction of DNA with Phenol/Chloroform>
Consult the protocol

2015.08.12

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
     1. Miniprep of p426Gal-Lucy-rho-CXCR1
  2. Electrophoresis to confirm whether transformed successfully or not
  3. Digestion of p426Gal-Lucy-rho-CXCR1
  4. Electrophoresis to confirm which colony been transformed successlly
  5. Transform Gpa1 PCR product into FAR1::KANMX-FUS1-GFP strain
< Miniprep of p426Gal-Lucy-rho-CXCR1>
Consult the protocol

< Electrophoresis to confirm whether transformed successfully or not>
Material: DNA marker: 1kb ladder 6ul
DNA sample: plasmid 2ul + 3ul 6x loading buffer
Condition: 0.5xTBE buffer 100V
Time: 40min
Result:

Note: This gel result shows size difference of plasmid transformed or untransformed.

< Digestion of p426Gal-Lucy-rho-CXCR1>

DNA(p426Gal-Lucy-rho-CXCR1 #2 #3 #7)	1μl
BamHI	0.5μl
XhoI	>0.5μl
10x NEB buffer #4	2.5μl
ddH2O	20.5μl
Total volume	25μl

Condition: incubate at 37℃ for 1hr

< Electrophoresis to confirm which colony been transformed successlly>
Material: DNA marker: 1kb ladder 6μl
DNA sample: plasmid 5μl + 3μl 6x loading buffer
Condition: 0.5xTBE buffer 100V
Time: 30min
Result:

Note: We use BamHI and XhoI to check whether the construct was built successfully.

< Transform Gpa1 PCR product into FAR1::KANMX-FUS1-GFP strain>
Consult the protocol
Yeast strain: FAR1::KANMX-FUS1-GFP
Selection plate : YPD+G418 plate

2015.0.

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
1. Digestion of pSB1C3 BBa_J04450 2. Electrophoresis to check digestion product 3. Digestion of pSB1C3 BBa_J04450 and rho-CXCR1 4. Gel extraction of vector and clean up insert 5. Ligation and transformation Experiment steps:

	Both	Only XbaI	Only SpeI	Uncut
pSB1C3 BBa_J04450#3	1μl	1μl	1μl	1μl
10x NEB buffer #4	2.5μl	2.5μl	2.5μl	0μl
XbaI	0.3μl	0.3μl	0μl	0μl
SpeI	0.3μl	0μl	0.3μl	0μl
ddH2O	21μl	21.2μl	21.2μl	24μl
Total volume	25μl	25μl	25μl	25μl

Condition: at 37℃ fpr 1hr < Electrophoresis to check digestion product> Material: DNA marker: 1kb ladder 6μl DNA sample: 10μl + 3μl 6x loading buffer Condition: 0.5xTBE buffer 100V Time: 30min Result: Note: It was up to our expection.

	Rho-CXCR1(400ng)	BBa_J04450(2μg)x2 tubes
DNA	8μl	9.6μl
10x NEB buffer#4	5μl	5μl
XbaI	1μl	2μl

2015.0.

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:

2015.0.

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:

2015.0.

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:

Toehold Switch As RNA Sensor

2015.6.20~2015.6.25

A, IL8 ; B, IL1β ; C, DUSP1 (dual specificity phosphatase 1) ;
D, SAT (spermidine/spermine N1-acetyltransferase EST

We use the website RNA structure to predict the secondary structure of these four toehold switches whether would have hairpin structures we want. Luckily, all of its results are match our expectation.

2015.6.25

Sent our designed sequences to IDT.

2015.7.1~2015.7.14

Amplify parts DNA we need.

2015.7.15

Receive IDT DNA.

2015.7.27

Transform BBa_I712019.

2015.7.28

Small scale incubation.

2015.7.29

Mini prep BBa_I712019 (Luciferase).
Concentration=186.2 (ng/ μl)

2015.7.30

Digest 4 toehold switches with EcoRI and XbaI and 4 trigger DNAs with XbaI and SpeI.


Plasmid DNA (25 ng/ μl)	5 μl
NEB #2	1 μl
EcoRI (20 U/μl)	0.2 μl
XbaI (20 U/μl	0.2 μl
ddH2O	3.6 μl
Total volume	10 μl

Incubate at 37˚C for 3 hr.


Plasmid DNA ( 25 ng/ μl))	5 μl
NEB #2	1 μl
EcoR I (20 U/μl)	0.2 μl
SpeI (20 U/μl)	0.2 μl
ddH2O	3.6 μl
Total volume	10 μl

Incubate at 37˚C for 3 hr.

2015.7.31

Do the electrophoresis of digested IDT products and gel extraction Restriction enzyme digestion of synthetic toehold and trigger RNA with EcoRI and SpeI. Samples were run in 1% agarose gel. Lane 1, 1kb DNA marker；Lane 2-5, 125 ng of synthetic DNA fragment that would transcribe into toehold sensors (SAT、DUSP1、IL-1β、IL-8 ) were digested by EcoRI and SpeI；Lane 6-9, 125 ng of DNA fragment that would transcribe into trigger RNAs (SAT、DUSP1、IL-1β、IL-8 ) were digested by EcoRI and SpeI.
All the results are around 100 bp, are the same as our expectation.

2015.8.3

Ask NYMU_Taiwan team for confirmed BBa_I712019, because we mistook the wrong antibiotic and lose all the parts DNA.

2015.8.4

Get the parts and transform parts BBa_I712019.


Vector DNA	2 μl
Competent cell (DH10β)	100 μl

2015.8.5

Do small scale incubation.

2015.8.6

MIniprep BBa_I712019.

2015.8.10

Small scale parts plasmids BBa_I712019 (Luciferase) digestion


Plasmid DNA (25 ng/ μl))	2.5 μl
NEB #2	1 μl
EcoRI (20 U/μl)	0.2 μl
SpeI (20 U/μl)	0.2 μl
ddH2O	6.1 μl
Total volume	10 μl

Incubate at 37˚C for 3 hr.

2015.8.11

Do the electrophoresis of BBa_I712019 (Luciferase) to check the size and condition for experiment and gel extraction to check the size.

Mini-prep trigger RNAs in pSB1AK8 backbone (total 20 tubes).

2015.8.12

Largely cut luciferase in pSB1AK8 and purify it, then we got linear luciferase.


Plasmid DNA (142.5 ng/ μl)	3 μl
Cut smart	3 μl
XbaI (20 U/μl)	0.6 μl
EcoRI (20 U/μl)	0.6 μl
ddH2O	22.8 μl
Total volume	30 μl

Incubate at 37˚C for 3 hr.
Lane 1 and 4, 3 μg of luciferase digested by EcoRI and XbaI ; Lane 2, 1K DNA marker ; Lane 3, 3 μg of luciferase not digested by EcoRI and XbaI.In the picture, uncut plasmid is higher than the cut ones. After discussing with our advisor, we suppose that the uncut plasmid is not functional fold, so it runs slower than the plasmid with digestion.

Ligation toehold switches with luciferase (pSB1AK8).

Ligation trigger RNAs into pSB1AK8 backbone.

2015.8.13

Transform two kinds of construct into DH10α bacteria respectively to do mass culture.

2015.8.14

Sent our E. coli and primer (VF2) to sequence for check our assembled result.

2015.8.17

Have synthetic toeholds (DUSP1, SAT, IL-8) -luciferase in pSB1AK8 sequence be confirmed.

2015.8.18

Small scale T7 promoter digestion.

2015.8.19

Large scale T7 promoter digestion, and gel extraction.


Plasmid DNA	30 μl
NEB #2	5 μl
PstI (20 U/μl)	3 μl
SpeI (20 U/μl)	2.6 μl
ddH2O	10 μl
Total volume	50 μl

Incubate at 37˚C for 3 hr.
Lane 1, marker ; Lane 2, 3 μg T7 promoter digested by restriction enzyme PstI and SpeI.

2015.8.20

Toehold+luciferase RNA enzyme digestion, gel extraction.


SAT1	241.7 (ng/ μl)
DUSP	236.2 (ng/ μl)
IL8	181.2(ng/ μl)

2015.8.21

Ligation trigger RNA to T7 promoter in pSB1AK8 backbone.


T7 promoter linear (119.6 ng/ μl)	15 μl
Plasmid DNA	2 μl
NEB #2	1.5 μl
PstI (20 U/μl)	3 μl
SpeI (20 U/μl)	3 μl
ddH2O	5.5 μl
Total volume	30 μl

The trigger RNA construct we finally complete. The structure contain T7 promoter, trigger, also Ampicillin antibiotic sequence easy for us to do the selection.

2015.8.22

Synthetic toehold–luciferase (IL1β) sequences confirmation.

T7 promoter-trigger RNA (SAT) in pSB1AK8 backbone sequences confirmation success, but T7 promoter-trigger RNA (DUSP1, IL1β,IL8) in pSB 1AK8 backbone sequences confirmation failed.

2015.8.23

Reconstruct the T7 promoter-trigger RNA (IL8,IL1 β,DUSP) in pSB1AK8 backbone.

2015.8.24

T7 promoter-trigger RNA(DUSP1, IL-1β,IL-8) in pSB1AK8 quick screen 6 colonies each.
1. Add100 μl lysis buffer into Eppendorf tube.
2. Use stick to take a little bit bacteria into lysis buffer.
3. Add 1:1 phenol/chloroform and shake it.
4. Centrifuge(12000g, 5mins).
5. Take 10μl to electrophoresis.

2015.8.25

Toehold switches digestion and ligation with pSB1C3 backbone.


Plasmid DNA (SAT1)	45 μl
NEB#2	6 μl
EcoRI (20 U/μl)	4 μl
PstI (20 U/μl)	4 μl
ddH2O	1 μl
Total volume	60 μl


Plasmid DNA (DUSP1)	45 μl
NEB#2	6 μl
EcoRI (20 U/μl	4 μl
PstI (20 U/μl	4 μl
ddH2O	1 μl
Total volume	60 μl


Plasmid DNA (IL8)	45 μl
NEB#2	6 μl
EcoRI (20 U/μl	4 μl
PstI (20 U/μl	4 μl
ddH2O	1 μl
Total volume	60 μl


Plasmid DNA (IL1β)	45 μl
NEB#2	6 μl
EcoRI (20 U/μl	4 μl
PstI (20 U/μl	4 μl
ddH2O	1 μl
Total volume	60 μl


BBa_I712074 linear	5 μl
Toehold switch DNA	4 μl
T4 ligase	1 μl
PEG400	2 μl
Total volume	20 μl

The construct of toehold switches part we done. The structure include T7 promoter, toehold switches, and reporter gene—the luciferase. What’s more, the plasmids contain the chloramphenicol resistance gene for selection.

2015.8.26

Transformation


Vector DNA (158 ng/μl)	1 μl
Competent cell (DH10β)	100 μl

2015.8.27

Pick single colony and do the quick screen to check if the parts are inserted.

2015.8.28

Small scale incubation.

2015.8.31

Do the miniprep.
DNA Concentration= 174ng/μl

2015.9.1

Cotransformation (DUSP1, IL-8, SAT) in the condition of Amp, CAM, Amp+CAM.

2015.9.2

T7-trigger (IL-1 β) ligation. In the figure, each lane are 10 μl of PCR products, we use temperature gradient (50°C, 53°C, 56°C, 59°C, 62°C, 65°C) to test the product of which temperature condition is suitable.
we choose the result of 53 °C for PCR. Because the product of 53 °C condition is much clearer, also much brighter then other conditions.

2015.9.3

Cotransformation (DUSP1, IL-8, SAT) in the condition of Amp+CAM .


Toehold plasmid	100 ng
Trigger plasmid	100 ng
Competent cell (BL21DE3)	100 μl

2015.9.6

1. Grow the overnight culture before the day of experiment.
2. Dilute 200 μl overnight culture with 10 ml LB broth and incubate for 2 hours.
3. Control the culture O.D between 0.6~0.8.
4. Spin down(6000g,3 mins) 9 ml culture in Eppendorf tube.
5. Wash the pellet with 1 ml PBS and spin down (6000 g, 3 min).
6. Add 500μl 1x luciferase passive lysis buffer and move to new mini beads beater tube with enough mini beads.
7. Put the tube into mini beads beater and shock 40 sec/time.
8. Shock 4~6 times and 2 mins on ice between every shock.
9. Spin down(12000g, 5mins)the lysate and move the supernatant into new Eppendorf tube.
10. Add 1ml 1X protein assay dye into cuvette and mix with 1μl lysate sample.
11. Put the sample into DU800 spectrometer and get the concentration data.
12. Normalize the quantity of protein(0.72 mg/ml) in each Eppendorf tube.
13. Add 50μl luciferin to each sample and put into GloMax® luminometer.
14. Get the number of luciferase activity.

2015.9.7

Cotransformation (DUSP1, IL-8, SAT) in the condition of Amp+CAM again to have another plate of PCR T7-luciferase (positive).


Toehold plasmid	100 ng
Trigger plasmid	100 ng
Competent cell (BL21DE3)	100 μl

@@ Line 1,195: / Line 1,195: @@
 														</div>
 													</div>
+													<div class="panel panel-default">
+														<div class="panel-heading">
+															<h4 class="panel-title">
+															<a data-toggle="collapse" data-parent="#accordion" href="#collapse">2015.0.</a>
+															</h4>
+														</div>
+														<div id="collapse" class="panel-collapse collapse ">
+															<div class="panel-body">
+																Operator: Wan-Yun, Jin-Ting, Jin-Ye<br>
+																Goal: <br>
+																&nbsp;&nbsp;1. Digestion of pSB1C3 BBa_J04450
+																&nbsp;&nbsp;2. Electrophoresis to check digestion product
+																&nbsp;&nbsp;3. Digestion of pSB1C3 BBa_J04450 and rho-CXCR1
+																&nbsp;&nbsp;4. Gel extraction of vector and clean up insert
+																&nbsp;&nbsp;5. Ligation and transformation
+																Experiment steps:
+																<Digestion of pSB1C3 BBa_J04450>
+																<table class="protocol-table">
+																	<thead>
+																		<tr><th>&nbsp;</th><th>Both</th><th>Only XbaI</th><th>Only SpeI</th><th>Uncut</th></tr>
+																	</thead>
+																	<tr><td>pSB1C3 BBa_J04450#3 </td><td>1μl</td><td>1μl</td><td>1μl </td><td>1μl</td></tr>
+																	<tr><td>10x NEB buffer #4 </td><td>2.5μl</td><td>2.5μl</td><td>2.5μl</td><td>0μl</td></tr>
+																	<tr><td>XbaI </td><td>0.3μl</td><td>0.3μl</td><td>0μl</td><td>0μl</td></tr>
+																	<tr><td>SpeI </td><td>0.3μl</td><td>0μl</td><td>0.3μl</td><td>0μl</td></tr>
+																	<tr><td>ddH2O </td><td>21μl</td><td>21.2μl</td><td>21.2μl</td><td>24μl </td></tr>
+																	<tr><td>Total volume </td><td>25μl </td><td>25μl </td><td>25μl </td><td>25μl </td></tr>
+																</table>
+																Condition:  at 37℃ fpr 1hr
+																< Electrophoresis to check digestion product>
+																Material: DNA marker: 1kb ladder 6μl
+																DNA sample: 10μl + 3μl 6x loading buffer
+																Condition: 0.5xTBE buffer 100V
+																Time: 30min
+																Result:
+																<img>
+																Note: It was up to our expection.
+																<Digestion of pSB1C3 BBa_J04450 and rho-CXCR1>
+																<table class="protocol-table">
+																	<thead>
+																		<tr><th>&nbsp;</th><th>Rho-CXCR1(400ng)</th><th>BBa_J04450(2μg)x2 tubes</th></tr>
+																	</thead>
+																	<tr><td>DNA </td><td>8μl</td><td>9.6μl </td></tr>
+																	<tr><td>10x NEB buffer#4</td><td>5μl </td><td>5μl</td></tr>
+																	<tr><td>XbaI </td><td> 1μl</td><td>		2μl </td></tr>
+																	<tr><td> </td><td> </td><td> </td></tr>
+																	<tr><td> </td><td> </td><td> </td></tr>
+																	<tr><td> </td><td> </td><td> </td></tr>
+																</table>
+															</div>
+														</div>
+													</div>
+													<div class="panel panel-default">
+														<div class="panel-heading">
+															<h4 class="panel-title">
+															<a data-toggle="collapse" data-parent="#accordion" href="#collapse">2015.0.</a>
+															</h4>
+														</div>
+														<div id="collapse" class="panel-collapse collapse ">
+															<div class="panel-body">
+																Operator: Wan-Yun, Jin-Ting, Jin-Ye<br>
+																Goal: <br>
+																&nbsp;&nbsp;
+															</div>
+														</div>
+													</div>
+													<div class="panel panel-default">
+														<div class="panel-heading">
+															<h4 class="panel-title">
+															<a data-toggle="collapse" data-parent="#accordion" href="#collapse">2015.0.</a>
+															</h4>
+														</div>
+														<div id="collapse" class="panel-collapse collapse ">
+															<div class="panel-body">
+																Operator: Wan-Yun, Jin-Ting, Jin-Ye<br>
+																Goal: <br>
+																&nbsp;&nbsp;
+															</div>
+														</div>
+													</div>
+													<div class="panel panel-default">
+														<div class="panel-heading">
+															<h4 class="panel-title">
+															<a data-toggle="collapse" data-parent="#accordion" href="#collapse">2015.0.</a>
+															</h4>
+														</div>
+														<div id="collapse" class="panel-collapse collapse ">
+															<div class="panel-body">
+																Operator: Wan-Yun, Jin-Ting, Jin-Ye<br>
+																Goal: <br>
+																&nbsp;&nbsp;
+															</div>
+														</div>
+													</div>
 <!--日期最小單位-->

Difference between revisions of "Team:CGU Taiwan/standard content"

Revision as of 18:07, 18 November 2015

Lab Note

Yeast With IL-8 Receptor

Toehold Switch As RNA Sensor