Team:Stockholm/4 October 2010

From 2010.igem.org

(Difference between revisions)

Latest revision as of 21:05, 27 October 2010

Andreas

Transfer of pEX.nCPP⋅SOD⋅His to BL21

Gel verification

Colony PCR gel verification of BL21 clones carrying pEX.nLMWP⋅SOD⋅His (1) and pEX.nTra10⋅SOD⋅His (2) plasmids.
4 μl λ; 5 μl sample;
λ = O'GeneRuler 1 kb DNA ladder.

Re-run of 2/10 BL21 samples

BL21 pEX.nLMWP⋅SOD⋅His: A & B
BL21 pEX.nTra10⋅SOD⋅His: A & B

1 % agarose, 120 V

Expected bands:

744 bp
765 bp

Results

Both clones verified
Clone A verified; weak band for clone B

ON cultures

3 ml LB, 30 °C
1. A (BL21 pEX.nLMWP⋅SOD⋅His)
2. A (BL21 pEX.nTra10⋅SOD⋅His)

Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX

Colony PCR

Picked 2 new colonies of each of the two constructs transformed 30/8:

5. pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: A & B
6. pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: A & B

Standard colony PCR settings

Elongation time: 2:00

Gel verification

Colony PCR gel verification of pEX.nCPP⋅SOD⋅His.RBS.yCCS operon clones.
4 μl λ; 5 μl sample;
λ = O'GeneRuler 1 kb DNA ladder.

0.8 % agarose, 100 V

Expected bands:

5. 1553 bp
6. 1553 bp

Results

5. Relevant band for clone B; too large insert (double?) for clone A.
6. Relevant band for clone B; too large insert (double?) for clone A.

ON cultures

5 ml LB, 37 °C, 250 rpm
- pEX.nTAT⋅SOD⋅His.RBS.yCCS 2: A
- pEX.nTAT⋅SOD⋅His.RBS.yCCS 3: B
- pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: B
- pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: B
- pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2: B
- pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3: B

Verification of pSB1x3 plasmids

Due to some strange growth results with our stock plasmids (pSB1x3.BBa_J04450), I decided to verify their antibiotic resistance. Restreaked clones of the following plasmids (w/ BBa_J04450 inserts) onto Amp 100, Km 50 and Cm 25 plates:

pSB1A3
pSB1C3
pSB1K3
pSB1AC3
pSB1AK3

Assembly of His⋅SOD⋅cCPP constructs

Continued from 1/10

Received cCPPs (cTra10, cTAT and cLMWP) in pSB1C3 plasmids, digested with EcoRI and NgoMIV, from Johan.

Ligations

Vectors:
1. Dig pSB1C3.cTra10 E+N
2. Dig pSB1C3.cTAT E+N
3. Dig pSB1C3.cLMWP E+N
Insert: Dig pMA.His⋅SOD E+A

	1	2	3
10X T4 Ligase buffer	2	2	2
Vector DNA	1	1	1
Insert DNA	5	5	5
dH₂O	11	11	11
T4 DNA ligase	1	1	1
	20 μl	20 μl	20 μl

Incubation: 22 °C, 15 min

Transformations

pSB1C3.His⋅SOD⋅cTra10
pSB1C3.His⋅SOD⋅cTAT
pSB1C3.His⋅SOD⋅cLMWP

Standard transformation
- 1 μl
- Cm 25

Nina

Mini prep on IgG_Tra10_N#6

I performed a mini prep on yesterday's inoculated IgG_Tra10_N colony # 6 in 12 ml LB with 24 ul chloramphenicol. The procedure was according to the method described in protocols.

Overday culture of SOD

I put an overday culture of SOD-peX, His-SOD, SOD-His, SOD_TAT_Ntermin and as a positive control yCC-peX from Andrea's glycerol stocks in 12 ml LB with 24 ul amphicillin. I inoculated with a big pipett tip amount in order to have the culture reaching OD 0.6 during the day of laboration.

The reason for why I did this culture was because I had talked to Mimmi about her overexpressions about these constructs and she hadn't got any satisfying results on her gels beside from the yCC sample (which became my positive control). I wanted therefore to check if I would also obtain her type of outcome when overexpressing the proteins of interest.

Gene digestions

I performed digestions of my genes of interest in order to do a gel clean up and follow with a ligation that should yield many positive cloning results.

Digestions:

Fusion_EA_His # 1 & 3 10 ul
Fast digestion buffer 10 X 2 ul
H2O 6 ul
Restriction enzyme NgoMVI 1 ul
Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C)

Fusion_NS_His # 2 10 ul
Fast digestion buffer 10 X 2 ul
H2O 6 ul
Restriction enzyme AgeI 1 ul
Restriction enzyme EcoRI 1 ul (Added after 1.5 h in 37 °C)

Protein A_EA_His # 5 10 ul
Fast digestion buffer 10 X 2 ul
H2O 6 ul
Restriction enzyme NgoMVI 1 ul
Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C)

IgG protease_EA_His # 5 10 ul
Fast digestion buffer 10 X 2 ul
H2O 6 ul
Restriction enzyme NgoMVI 1 ul
Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C)

IgG protease_Tra10_Ntermin # 4 & 6 10 ul
Fast digestion buffer 10 X 2 ul
H2O 6 ul
Restriction enzyme XbaI 1 ul
Restriction enzyme PstI 1 ul

Incubate in 37 °C for 30 minutes.

Agarose gel on digests

I ran an agarose gel 1 % 100 V on the digested samples in order to check if they have been digested by the enzymes and followed by cutting the bands of interest out of the gel with a scalpel over a UV-lamp for a gel clean up.

Ladder: MassRuler™ DNA Ladder Mix, ready-to-use, 80-10,000 bp

Arrangement on gels:

Gel clean up

I performed a gel clean up of the digested genes. All except IgGEA#5 were digested and cut out of the gel.

I performed the clean up according to the method described in protocols.

The measurements of the cut gel bands, addition of kit solutions and incubation time:

All bands had a weight of aproximately 0.12 g (120 mg). 120 mg * 3 = 360 ul QXI

I added 10 ul of QIAEXII to all samples

All samples were incubated for 5 minutes at RT

I performed step 11 in the procedure description

Concentration measurments

I measured with a spectrophotometer the concentration of the gel clean up samples.

Sending for sequencing

I sent IgG_Tra10_Ntermin#6 for sequencing. I mixed 15 ul of the miniprep sample and 1.5 ul of Forward bank vector verification primer (VF).

ASB0045 898

Mimmi

Protein purification

Using Qiagen Ni-NTA Spin Kit

Buffers

Lysis buffer NPI-10

Wash buffer NPI-20

NaH₂PO₄•H₂O

NaCl

+ imidazole

3.45g

8.77g

6.8g

50mM

300mM

2M

\

/ 500ml

100ml

Elution buffer

NaH₂PO₄•H₂O

NaCl

imidazole

3.45g

8.77g

17g

50mM

300mM

500mM

)

> 500ml

)

Lysosyme

0.1g

10mg/ml

10ml

Johan

Miniprep

tra10-bFGF-his, tat-bFGF-his, tat-bFGF-his, lmwp-bFGF-his, his-bFGF-tra10, his-bFGF-lmwp

All ~300 ng/µl

Digest miniprep

2 µl DNA

(1 µl BamHI)

2 µl 10x buffer

15 µl H2O

Gel

tra10-bFGF-his, tat-bFGF-his, tat-bFGF-his, lmwp-bFGF-his, his-bFGF-tra10, his-bFGF-lmwp

Results: All constructs had one correct miniprep

Cut

The parts was cut from its vector to be ligated into pEX vector

5 µl bFGF

2 µl 10x buffer

1 µl XbaI

1 µl PstI

11 µl H2O

Ligation

The vector was first treated with alkaline phosphatase for 60 min.

7 µl bFGF

1 µl pEX

2 µl 10x buffer

1 µl ligase

9 µl H2O

Transformation

3 µl from all constructs was then transformed into top10 cells.

Coomassie gels

I made 2 coomassie gels to be used tomorrow

The Faculty of Science at Stockholm University

Swedish Vitiligo association (Svenska Vitiligoförbundet)

@@ Line 1: / Line 1: @@
 {{Stockholm/Top2}}
 ==Andreas==
 ===Transfer of pEX.nCPP⋅SOD⋅His to BL21===
 ====Gel verification====
+[[image:ColPCR_BL21CPP_4oct.png|200px|thumb|right|'''Colony PCR gel verification of BL21 clones carrying pEX.nLMWP&sdot;SOD&sdot;His (1) and pEX.nTra10&sdot;SOD&sdot;His (2) plasmids.'''<br />4 &mu;l &lambda;; 5 &mu;l sample;<br />&lambda; = O'GeneRuler 1 kb DNA ladder.]]
 ''Re-run of 2/10 BL21 samples''
@@ Line 20: / Line 22: @@
 ====ON cultures====
 *3 ml LB, 30 &deg;C
-*# A: BL21 pEX.nLMWP&sdot;SOD&sdot;His
+*# A (BL21 pEX.nLMWP&sdot;SOD&sdot;His)
-*# A: BL21 pEX.nTra10&sdot;SOD&sdot;His
+*# A (BL21 pEX.nTra10&sdot;SOD&sdot;His)
 ===Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX===
@@ Line 33: / Line 35: @@
 ====Gel verification====
+[[image:ColPCR_CPP-SH-Ry_operon_4oct.png‎|200px|thumb|right|'''Colony PCR gel verification of pEX.nCPP&sdot;SOD&sdot;His.RBS.yCCS operon clones.'''<br />4 &mu;l &lambda;; 5 &mu;l sample;<br />&lambda; = O'GeneRuler 1 kb DNA ladder.]]
 .8 % agarose, 100 V
@@ Line 45: / Line 48: @@
 ====ON cultures====
 *5 ml LB, 37 &deg;C, 250 rpm
-** pEX.nTAT⋅SOD⋅His.RBS.yCCS 2: A & B
+** pEX.nTAT⋅SOD⋅His.RBS.yCCS 2: A
-** pEX.nTAT⋅SOD⋅His.RBS.yCCS 3: A & B
+** pEX.nTAT⋅SOD⋅His.RBS.yCCS 3: B
-** pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: A & B
+** pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: B
-** pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: A & B
+** pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: B
-** pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2: A & B
+** pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2: B
-** pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3: A & B
+** pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3: B
+===Verification of pSB1x3 plasmids===
+Due to some strange growth results with our stock plasmids (pSB1x3.BBa_J04450), I decided to verify their antibiotic resistance. Restreaked clones of the following plasmids (w/ BBa_J04450 inserts) onto Amp 100, Km 50 and Cm 25 plates:
+*pSB1A3
+*pSB1C3
+*pSB1K3
+*pSB1AC3
+*pSB1AK3
+===Assembly of His⋅SOD⋅cCPP constructs===
+''Continued from 1/10''
+Received cCPPs (cTra10, cTAT and cLMWP) in pSB1C3 plasmids, digested with EcoRI and NgoMIV, from Johan.
+====Ligations====
+*Vectors:
+*#Dig pSB1C3.cTra10 E+N
+*#Dig pSB1C3.cTAT E+N
+*#Dig pSB1C3.cLMWP E+N
+*Insert: Dig pMA.His&sdot;SOD E+A
+{|border="1" cellpadding="1" cellspacing="0"
+|&nbsp;
+!width="50"|1
+!width="50"|2
+!width="50"|3
+|-
+|10X T4 Ligase buffer
+|align="center"|2
+|align="center"|2
+|align="center"|2
+|-
+|Vector DNA
+|align="center"|1
+|align="center"|1
+|align="center"|1
+|-
+|Insert DNA
+|align="center"|5
+|align="center"|5
+|align="center"|5
+|-
+|dH<sub>2</sub>O
+|align="center"|11
+|align="center"|11
+|align="center"|11
+|-
+|T4 DNA ligase
+|align="center"|1
+|align="center"|1
+|align="center"|1
+|-
+|&nbsp;
+!20 &mu;l
+!20 &mu;l
+!20 &mu;l
+|}
+*Incubation: 22 &deg;C, 15 min
+====Transformations====
+#pSB1C3.His&sdot;SOD&sdot;cTra10
+#pSB1C3.His&sdot;SOD&sdot;cTAT
+#pSB1C3.His&sdot;SOD&sdot;cLMWP
+*Standard transformation
+**1 &mu;l
+**Cm 25
+----
+----
+==Nina==
+===Mini prep on IgG_Tra10_N#6===
+I performed a mini prep on yesterday's inoculated IgG_Tra10_N colony # 6 in 12 ml LB with 24 ul chloramphenicol. The procedure was according to the method described in protocols.
+===Overday culture of SOD===
+I put an overday culture of SOD-peX, His-SOD, SOD-His, SOD_TAT_Ntermin and as a positive control yCC-peX from Andrea's glycerol stocks in 12 ml LB with 24 ul amphicillin. I inoculated with a big pipett tip amount in order to have the culture reaching OD 0.6 during the day of laboration.
+The reason for why I did this culture was because I had talked to Mimmi about her overexpressions about these constructs and she hadn't got any satisfying results on her gels beside from the yCC sample (which became my positive control). I wanted therefore to check if I would also obtain her type of outcome when overexpressing the proteins of interest.
+===Gene digestions===
+I performed digestions of my genes of interest in order to do a gel clean up and follow with a ligation that should yield many positive cloning results.
+Digestions:
+*Fusion_EA_His # 1 & 3 10 ul
+*Fast digestion buffer 10 X 2 ul
+*H2O 6 ul
+*Restriction enzyme NgoMVI 1 ul
+*Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C)
+*Fusion_NS_His # 2 10 ul
+*Fast digestion buffer 10 X 2 ul
+*H2O 6 ul
+*Restriction enzyme AgeI 1 ul
+*Restriction enzyme EcoRI 1 ul (Added after 1.5 h in 37 °C)
+*Protein A_EA_His # 5 10 ul
+*Fast digestion buffer 10 X 2 ul
+*H2O 6 ul
+*Restriction enzyme NgoMVI 1 ul
+*Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C)
+*IgG protease_EA_His # 5 10 ul
+*Fast digestion buffer 10 X 2 ul
+*H2O 6 ul
+*Restriction enzyme NgoMVI 1 ul
+*Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C)
+*IgG protease_Tra10_Ntermin # 4 & 6 10 ul
+*Fast digestion buffer 10 X 2 ul
+*H2O 6 ul
+*Restriction enzyme XbaI 1 ul
+*Restriction enzyme PstI 1 ul
+Incubate in 37 °C for 30 minutes.
+===Agarose gel on digests===
+I ran an agarose gel 1 % 100 V on the digested samples in order to check if they have been digested by the enzymes and followed by cutting the bands of interest out of the gel with a scalpel over a UV-lamp for a gel clean up.
+Ladder: MassRuler™ DNA Ladder Mix, ready-to-use, 80-10,000 bp
+[[Image:laddermixmassruler.jpg|200px]]
+Arrangement on gels:
+[[Image:Az.jpg]]
+===Gel clean up===
+I performed a gel clean up of the digested genes. All except IgGEA#5 were digested and cut out of the gel.
+[[Image:XD.jpg|250px]]
+I performed the clean up according to the method described in protocols.
+The measurements of the cut gel bands, addition of kit solutions and incubation time:
+*All bands had a weight of aproximately 0.12 g (120 mg). 120 mg * 3 = 360 ul QXI
+*I added 10 ul of QIAEXII to all samples
+*All samples were incubated for 5 minutes at RT
+*I performed step 11 in the procedure description
+===Concentration measurments===
+I measured with a spectrophotometer the concentration of the gel clean up samples.
+[[Image:Nm.jpg]]
+===Sending for sequencing===
+I sent IgG_Tra10_Ntermin#6 for sequencing. I mixed 15 ul of the miniprep sample and 1.5 ul of Forward bank vector verification primer (VF).
+* ASB0045 898
+== Mimmi ==
+=== Protein purification ===
+*Using Qiagen Ni-NTA Spin Kit
+'''Buffers'''
+{| border="1"
+| Lysis buffer NPI-10
+Wash buffer NPI-20
+| NaH<sub>2</sub>PO<sub>4</sub>&bull;H<sub>2</sub>O
+NaCl
++
+imidazole
+|3.45g
+.77g
+.8g
+| 50mM
+mM
+M
+| \
+/ 500ml
+ml
+|-
+|
+Elution buffer
+| NaH<sub>2</sub>PO<sub>4</sub>&bull;H<sub>2</sub>O
+NaCl
+imidazole
+|3.45g
+.77g
+g
+| 50mM
+mM
+mM
+| )
+> 500ml
+)
+|-
+| Lysosyme
+|
+| 0.1g
+| 10mg/ml
+| 10ml
+|}
+==Johan==
+===Miniprep===
+tra10-bFGF-his, tat-bFGF-his, tat-bFGF-his, lmwp-bFGF-his, his-bFGF-tra10, his-bFGF-lmwp
+All ~300 ng/µl
+===Digest miniprep===
+µl DNA
+(1 µl BamHI)
+µl 10x buffer
+µl H2O
+===Gel===
+tra10-bFGF-his, tat-bFGF-his, tat-bFGF-his, lmwp-bFGF-his, his-bFGF-tra10, his-bFGF-lmwp
+[[Image:SU 4oktgels.png]]
+Results: All constructs had one correct miniprep
+===Cut===
+The parts was cut from its vector to be ligated into pEX vector
+µl bFGF
+µl 10x buffer
+µl XbaI
+µl PstI
+µl H2O
+===Ligation===
+The vector was first treated with alkaline phosphatase for 60 min.
+µl bFGF
+µl pEX
+µl 10x buffer
+µl ligase
+µl H2O
+===Transformation===
+µl from all constructs was then transformed into top10 cells.
+===Coomassie gels===
+I made 2 coomassie gels to be used tomorrow
+{{Stockholm/Footer}}

Team:Stockholm/4 October 2010

From 2010.igem.org

Latest revision as of 21:05, 27 October 2010

Contents

Andreas

Transfer of pEX.nCPP⋅SOD⋅His to BL21

Gel verification

ON cultures

Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX

Colony PCR

Gel verification

ON cultures

Verification of pSB1x3 plasmids

Assembly of His⋅SOD⋅cCPP constructs

Ligations

Transformations

Nina

Mini prep on IgG_Tra10_N#6

Overday culture of SOD

Gene digestions

Agarose gel on digests

Gel clean up

Concentration measurments

Sending for sequencing

Mimmi

Protein purification

Johan

Miniprep

Digest miniprep

Gel

Cut

Ligation

Transformation

Coomassie gels