Team:Stockholm/29 September 2010

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==Andreas==
==Andreas==
 +
 +
===Sequencing results===
 +
Sequencing results from 27/9 samples arrived and were analyzed by nucleotide BLAST.
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!Construct
 +
!Sequencing<br />result
 +
!Blastn<br />result
 +
!Comment
 +
|-
 +
|pEX.N-TAT&sdot;SOD&sdot;His 3
 +
|[[media:PEX.nTAT.SOD.his3_premix_fasta_29sep.txt|pEX.nTAT.SOD.his 3_premix]]
 +
|[[media:Blastn_pEX.nTAT.SOD.his_3_premix_29sep.txt‎|Blastn]]
 +
|'''<span style="color:green">Verified'''</span>. Three silent mutations in His tag; does not alter a.a. sequence.
 +
|-
 +
|pEX.N-TAT&sdot;SOD&sdot;His 4
 +
|[[media:PEX.nTAT.SOD.his4_premix_fasta_29sep.txt‎|pEX.nTAT.SOD.his 4_premix]]
 +
|[[media:Blastn_pEX.nTAT.SOD.his_4_premix_29sep.txt‎|Blastn]]
 +
|'''<span style="color:red;">Not verified</span>'''. Incorrect construct.
 +
|-
 +
|pSB1A2.RBS.yCCS 3
 +
|[[media:PSB1A2.RBS.yCCS3_premix_fasta_29sep.txt|pSB1A2.RBS.yCCS 3_premix]]
 +
|[[media:Blastn_pSB1A2.RBS.yCCS3_premix_29sep.txt|Blastn]]
 +
|'''<span style="color:orange">Partly verified</span>'''. Three mutations found: (1) Single base deletion in yCCS sequence, causing a frame shift and an extended open reading frame; (2) Insertion between SpeI and PstI, not affecting expression of cloning; (3) Point mutation in PstI site, disabling PstI restriction. Mutations (1) and (3) are both quite unsure, as the sequencing quality in this area is very low. New sequencing from VR will be sent.
 +
|-
 +
|pSB1A2.RBS.yCCS 4
 +
|[[media:PSB1A2.RBS.yCCS4_premix_fasta_29sep.txt|pSB1A2.RBS.yCCS 4_premix]]
 +
|[[media:Blastn_pSB1A2.RBS.yCCS4_premix_29sep.txt|Blastn]]
 +
|'''<span style="color:orange">Partly verified</span>'''. Four mutations found: (1) Point mutation changing a K codon to T; (2) Single base deletion in yCCS sequence, causing frame shift; (3) Insertion between SpeI and PstI, not affecting expression of cloning; (4) Point mutation in PstI site, disabling PstI restriction. See comment
 +
|-
 +
|pSB1K3.N-TAT&sdot;SOD&sdot;His 4
 +
|[[media:Blastn_pSB1K3.nTAT.SOD.his4_premix_29sep.txt|pSB1K3.nTAT.SOD.his4_premix]]
 +
|[[media:Blastn_pSB1K3.nTAT.SOD.his4_premix_29sep.txt|Blastn]]
 +
|'''<span style="color:green">Verified</span>'''. Three silent mutations in His tag; does not alter a.a. sequence.
 +
|-
 +
|pSB1K3.N-TAT&sdot;SOD&sdot;His 5
 +
|[[media:Blastn_pSB1K3.nTAT.SOD.his5_premix_29sep.txt|pSB1K3.nTAT.SOD.his5_premix]]
 +
|[[media:Blastn_pSB1K3.nTAT.SOD.his5_premix_29sep.txt|Blastn]]
 +
|'''<span style="color:green">Verified</span>'''. Three silent mutations in His tag; does not alter a.a. sequence.
 +
|-
 +
|pSB1K3.N-Tra10&sdot;SOD&sdot;His 5
 +
|[[media:PSB1K3.nTra10.SOD.his5_premix_fasta_29sep.txt|pSB1K3.nTra10.SOD.his5_premix]]
 +
|[[media:Blastn_pSB1K3.nTra10.SOD.h5_premix_29sep.txt‎|Blastn]]
 +
|'''<span style="color:green">Verified</span>'''. Three silent mutations in His tag; does not alter a.a. sequence.
 +
|-
 +
|-
 +
|pSB1K3.N-LMWP&sdot;SOD&sdot;His 1
 +
|[[media:PSB1K3.nLMWP.SOD.his1_premix_fasta_29sep.txt|pSB1K3.nLMWP.SOD.his1_premix]]
 +
|[[media:Blastn_pSB1K3.nLMWP.SOD.hi1_premix_29sep.txt|Blastn]]
 +
|'''<span style="color:green">Verified</span>'''. Three silent mutations in His tag; does not alter a.a. sequence.
 +
|-
 +
|-
 +
|pSB1C3.N-LMWP&sdot;SOD&sdot;His 1
 +
|[[media:PSB1C3.nLMWP.SOD.his1_premix_fasta_29sep.txt|pSB1C3.nLMWP.SOD.his1_premix]]
 +
|[[media:Blastn_pSB1C3.nLMWP.SOD.hi1_premix_29sep.txt|Blastn]]
 +
|'''<span style="color:red">Not verified'''</span>. Incorrect construct.
 +
|-
 +
|pSB1C3.N-LMWP&sdot;SOD&sdot;His 4
 +
|[[media:PSB1C3.nLMWP.SOD.his4_premix_fasta_29sep.txt|pSB1C3.nLMWP.SOD.his4_premix]]
 +
|[[media:Blastn_pSB1C3.nLMWP.SOD.hi4_premix_29sep.txt|Blastn]]
 +
|'''<span style="color:red">Not verified'''</span>. Incorrect construct.
 +
|-
 +
|pEX.SOD
 +
|[[media:PEX.SOD_premix_fasta_29sep.txt‎|pEX.SOD_premix]]
 +
|[[media:Blastn_pEX.SOD_premix_29sep.txt|Blastn]]
 +
|'''<span style="color:orange">Partly verified'''</span>. Insertion found between SpeI and PstI; does not affect expression.
 +
|-
 +
|pEX.yCCS 5
 +
|[[media:PEX.yCCS_5_premix_fasta.txt|pEX.yCCS 5_premix]]
 +
|[[media:Blastn_pEX.yCCS_5_premix_29sep.txt|Blastn]]
 +
|'''<span style="color:orange">Partly verified'''</span>. Insertion found between SpeI and PstI; does not affect expression.
 +
|-
 +
|pEX.SOD&sdot;His
 +
|[[media:PEX.SOD.his_premix_fasta_29sep.txt|pEX.SOD.his_premix]]
 +
|[[media:Blastn_pEX.SOD.his_premix_29sep.txt|Blastn]]
 +
|'''<span style="color:green">Verified'''</span>. Three silent mutations in His tag; does not alter a.a. sequence.
 +
|-
 +
|pEX.His&sdot;SOD
 +
|[[media:PEX.his.SOD_premix_29sep.txt‎|pEX.his.SOD_premix]]
 +
|[[media:Blastn_pEX.his.SOD_premix_29sep.txt|Blastn]]
 +
|'''<span style="color:green">Verified'''</span>. Three silent mutations in His tag; does not alter a.a. sequence.
 +
|}
 +
 +
===Plasmid prep===
 +
''From 28/9 ON cultures''
 +
 +
Spun down ON cultures 10 min, 3000 x ''g''. Decanted supernatant and stored pellets in -20 &deg;C ON for later plasmid prep.
 +
 +
===Sorting glycerol stocks===
 +
Started sorting our glycerol stocks into a spreadsheet for future reference.
 +
 +
==Nina==
 +
 +
===Agarose gel verificaion===
 +
 +
I ran an agarose gel 1 % 100 V on the colony PCR products from yesterday to check if I had any inserts.
 +
 +
Ladder: MassRuler™ DNA Ladder Mix, ready-to-use, 80-10,000 bp
 +
 +
[[Image:laddermixmassruler.jpg|200px]]
 +
 +
Arrangement on gel:
 +
 +
[[Image:Aq20.jpg]]
 +
 +
From left to right: 1-5 fusion EA, 1-5 Fusion NS, 1-5 IgG LMWP, 1-5 IgG_TAT_N, 1-5 IgG_Tra10_N, 1-5 IgG_TAT_C, 1-5 Protein A_TAT_C, 1-5 pEX, 1-9 pEX
 +
 +
[[Image:Aq21.jpg|200px]]
 +
 +
[[Image:Aq22.jpg|200px]]
 +
 +
[[Image:Aq23.jpg|200px]]
 +
 +
===Colony PCR===
 +
 +
I ran a new colony PCR on the samples I did not have a possitive result on from the gel above.
 +
 +
The samples for this screen are:
 +
 +
IgG_LMWP_N, _TAT_N, _Tra10_N, protein A_LMWP_N_pex, _TAT_N_pex & _Tra10_N_pex
 +
 +
I screened 8 colonies/dish.
 +
 +
PCR master mix:
 +
 +
*MgCl2 8ul
 +
*phusion buffer 5X 80ul
 +
*dNTP 8ul
 +
*primerR 24ul
 +
*primerF 24ul
 +
*polymerase 8ul
 +
*H2O 240ul
 +
 +
===Agarose gel verificaion===
 +
 +
I ran an agarose gel 1 % 100 V on the colony PCR products from yesterday to check if I had any inserts.
 +
 +
Ladder: MassRuler™ DNA Ladder Mix, ready-to-use, 80-10,000 bp
 +
 +
[[Image:laddermixmassruler.jpg|200px]]
 +
 +
Arrangement on gel:
 +
 +
[[Image:Aq24.jpg]]
 +
 +
[[Image:Aq25.jpg|200px]]
 +
 +
[[Image:Aq26.jpg|200px]]
 +
 +
[[Image:Aq27.jpg|200px]]
 +
 +
===Overnight culture===
 +
 +
I inoculated Fusion EA colony 1 & 3 and Fusion NS colony 1 & 2 each in 12 ml LB with 24 ul chloramphenicol resistance.
 +
 +
===Digestion===
 +
 +
I digested IgG protease and protein A in the bank vector C in order to perform a gel clean up of the genes and insert them into a pMa-His AS vector.
 +
 +
Digestion:
 +
 +
*H2O 15 ul
 +
*DNA 2 ul
 +
*Fastdigest buffer 10X 2 ul
 +
*Restriction enzyme NgoMIV 1 ul
 +
*Restriction enzyme SpeI 1 ul (Add after 1.5h incubation in 37 °C)
 +
 +
 +
 +
== Mimmi ==
 +
 +
=== Over expression ===
 +
 +
*Load gel
 +
***dilute 3h samples 1:4
 +
***load 8µl in the pre-wells
 +
***the comb takes up 4µl to load on the gel
 +
***run PhastGel 20%
 +
 +
{|
 +
! well
 +
! sample
 +
| rowspan="6" | [[Image:Place_for_picture.jpg|100px|thumb|left|]]
 +
! well
 +
! sample
 +
| rowspan="6" | [[Image:Place_for_picture.jpg|100px|thumb|left|]]
 +
|-
 +
| 1
 +
| ladder
 +
| 1
 +
| ladder
 +
|-
 +
| 2
 +
| SOD 0h
 +
| 2
 +
| SOD.his 0h
 +
|-
 +
| 3
 +
| SOD 3h
 +
| 3
 +
| SOD.his 3h
 +
|-
 +
| 4
 +
| yCCS 0h
 +
| 4
 +
| his.SOD 0h
 +
|-
 +
| 5
 +
| yCCS 3h
 +
| 5
 +
| his.SOD 3h
 +
|}
 +
 +
==Johan==
 +
 +
===Cut CPP-vector===
 +
 +
5 µl vector ~1µg
 +
 +
1 µl NgoMIV
 +
 +
1 µl EcoRI
 +
 +
2 µl 10x fastbuffer
 +
 +
12 µl H2O
 +
 +
Then heat-inactivation of enzymes
 +
 +
===Cut his-bFGF ===
 +
 +
5 µl his-bFGF (1,5 µl -> 0,3 µg bFGF)
 +
 +
1 µl AgeI
 +
 +
1 µl EcoRI
 +
 +
2 µl 10x fastbuffer
 +
 +
12 µl H2O
 +
 +
Then heat-inactivation of enzymes
 +
 +
===Ligation his-bFGF into CPP-vector===
 +
 +
5 µl his-bFGF
 +
 +
0,5 µl cpp-vector
 +
 +
2 µl buffer
 +
 +
1 µl T4 ligase
 +
 +
11,5 µl H2O
 +
 +
===Transformation===
 +
 +
3 µl of all constructs was transformed into top10 cells
 +
 +
{{Stockholm/Footer}}

Latest revision as of 21:40, 27 October 2010

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Contents

Andreas

Sequencing results

Sequencing results from 27/9 samples arrived and were analyzed by nucleotide BLAST.

Construct Sequencing
result 		Blastn
result 		Comment
pEX.N-TAT⋅SOD⋅His 3 pEX.nTAT.SOD.his 3_premix Blastn Verified. Three silent mutations in His tag; does not alter a.a. sequence.
pEX.N-TAT⋅SOD⋅His 4 pEX.nTAT.SOD.his 4_premix Blastn Not verified. Incorrect construct.
pSB1A2.RBS.yCCS 3 pSB1A2.RBS.yCCS 3_premix Blastn Partly verified. Three mutations found: (1) Single base deletion in yCCS sequence, causing a frame shift and an extended open reading frame; (2) Insertion between SpeI and PstI, not affecting expression of cloning; (3) Point mutation in PstI site, disabling PstI restriction. Mutations (1) and (3) are both quite unsure, as the sequencing quality in this area is very low. New sequencing from VR will be sent.
pSB1A2.RBS.yCCS 4 pSB1A2.RBS.yCCS 4_premix Blastn Partly verified. Four mutations found: (1) Point mutation changing a K codon to T; (2) Single base deletion in yCCS sequence, causing frame shift; (3) Insertion between SpeI and PstI, not affecting expression of cloning; (4) Point mutation in PstI site, disabling PstI restriction. See comment
pSB1K3.N-TAT⋅SOD⋅His 4 pSB1K3.nTAT.SOD.his4_premix Blastn Verified. Three silent mutations in His tag; does not alter a.a. sequence.
pSB1K3.N-TAT⋅SOD⋅His 5 pSB1K3.nTAT.SOD.his5_premix Blastn Verified. Three silent mutations in His tag; does not alter a.a. sequence.
pSB1K3.N-Tra10⋅SOD⋅His 5 pSB1K3.nTra10.SOD.his5_premix Blastn Verified. Three silent mutations in His tag; does not alter a.a. sequence.
pSB1K3.N-LMWP⋅SOD⋅His 1 pSB1K3.nLMWP.SOD.his1_premix Blastn Verified. Three silent mutations in His tag; does not alter a.a. sequence.
pSB1C3.N-LMWP⋅SOD⋅His 1 pSB1C3.nLMWP.SOD.his1_premix Blastn Not verified. Incorrect construct.
pSB1C3.N-LMWP⋅SOD⋅His 4 pSB1C3.nLMWP.SOD.his4_premix Blastn Not verified. Incorrect construct.
pEX.SOD pEX.SOD_premix Blastn Partly verified. Insertion found between SpeI and PstI; does not affect expression.
pEX.yCCS 5 pEX.yCCS 5_premix Blastn Partly verified. Insertion found between SpeI and PstI; does not affect expression.
pEX.SOD⋅His pEX.SOD.his_premix Blastn Verified. Three silent mutations in His tag; does not alter a.a. sequence.
pEX.His⋅SOD pEX.his.SOD_premix Blastn Verified. Three silent mutations in His tag; does not alter a.a. sequence.

Plasmid prep

From 28/9 ON cultures

Spun down ON cultures 10 min, 3000 x g. Decanted supernatant and stored pellets in -20 °C ON for later plasmid prep.

Sorting glycerol stocks

Started sorting our glycerol stocks into a spreadsheet for future reference.

Nina

Agarose gel verificaion

I ran an agarose gel 1 % 100 V on the colony PCR products from yesterday to check if I had any inserts.

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

Laddermixmassruler.jpg

Arrangement on gel:

Aq20.jpg

From left to right: 1-5 fusion EA, 1-5 Fusion NS, 1-5 IgG LMWP, 1-5 IgG_TAT_N, 1-5 IgG_Tra10_N, 1-5 IgG_TAT_C, 1-5 Protein A_TAT_C, 1-5 pEX, 1-9 pEX

Aq21.jpg

Aq22.jpg

Aq23.jpg

Colony PCR

I ran a new colony PCR on the samples I did not have a possitive result on from the gel above.

The samples for this screen are:

IgG_LMWP_N, _TAT_N, _Tra10_N, protein A_LMWP_N_pex, _TAT_N_pex & _Tra10_N_pex

I screened 8 colonies/dish.

PCR master mix:

  • MgCl2 8ul
  • phusion buffer 5X 80ul
  • dNTP 8ul
  • primerR 24ul
  • primerF 24ul
  • polymerase 8ul
  • H2O 240ul

Agarose gel verificaion

I ran an agarose gel 1 % 100 V on the colony PCR products from yesterday to check if I had any inserts.

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

Laddermixmassruler.jpg

Arrangement on gel:

Aq24.jpg

Aq25.jpg

Aq26.jpg

Aq27.jpg

Overnight culture

I inoculated Fusion EA colony 1 & 3 and Fusion NS colony 1 & 2 each in 12 ml LB with 24 ul chloramphenicol resistance.

Digestion

I digested IgG protease and protein A in the bank vector C in order to perform a gel clean up of the genes and insert them into a pMa-His AS vector.

Digestion:

  • H2O 15 ul
  • DNA 2 ul
  • Fastdigest buffer 10X 2 ul
  • Restriction enzyme NgoMIV 1 ul
  • Restriction enzyme SpeI 1 ul (Add after 1.5h incubation in 37 °C)


Mimmi

Over expression

  • Load gel
      • dilute 3h samples 1:4
      • load 8µl in the pre-wells
      • the comb takes up 4µl to load on the gel
      • run PhastGel 20%
well sample
Place for picture.jpg
well sample
Place for picture.jpg
1 ladder 1 ladder
2 SOD 0h 2 SOD.his 0h
3 SOD 3h 3 SOD.his 3h
4 yCCS 0h 4 his.SOD 0h
5 yCCS 3h 5 his.SOD 3h

Johan

Cut CPP-vector

5 µl vector ~1µg

1 µl NgoMIV

1 µl EcoRI

2 µl 10x fastbuffer

12 µl H2O

Then heat-inactivation of enzymes

Cut his-bFGF

5 µl his-bFGF (1,5 µl -> 0,3 µg bFGF)

1 µl AgeI

1 µl EcoRI

2 µl 10x fastbuffer

12 µl H2O

Then heat-inactivation of enzymes

Ligation his-bFGF into CPP-vector

5 µl his-bFGF

0,5 µl cpp-vector

2 µl buffer

1 µl T4 ligase

11,5 µl H2O

Transformation

3 µl of all constructs was transformed into top10 cells





The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/

Retrieved from "http://2010.igem.org/Team:Stockholm/29_September_2010"