Team:Stockholm/4 October 2010

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 +
==Andreas==
==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''
 +
 +
#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 &deg;C
 +
*# A (BL21 pEX.nLMWP&sdot;SOD&sdot;His)
 +
*# A (BL21 pEX.nTra10&sdot;SOD&sdot;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====
 +
[[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.]]
 +
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 &deg;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:
 +
*#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
 +
8.77g
 +
 +
6.8g
 +
| 50mM
 +
300mM
 +
 +
2M
 +
| \
 +
/ 500ml
 +
 +
100ml
 +
|-
 +
|
 +
Elution buffer
 +
 +
| NaH<sub>2</sub>PO<sub>4</sub>&bull;H<sub>2</sub>O
 +
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
 +
 +
[[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
 +
 +
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
 +
 +
{{Stockholm/Footer}}

Latest revision as of 21:05, 27 October 2010

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Contents

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

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

1 % agarose, 120 V

Expected bands:

  1. 744 bp
  2. 765 bp

Results

  1. Both clones verified
  2. 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

  1. pSB1C3.His⋅SOD⋅cTra10
  2. pSB1C3.His⋅SOD⋅cTAT
  3. 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

Laddermixmassruler.jpg

Arrangement on gels:

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.

XD.jpg

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.

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

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

SU 4oktgels.png

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) Geneious Fermentas/ Sigma-Aldrich/

Retrieved from "http://2010.igem.org/Team:Stockholm/4_October_2010"