Team:Stockholm/9 September 2010

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==Andreas==
==Andreas==
 +
 +
===Cloning of N-CPPs into pSB1C3===
 +
Since we realized that the method we used for cloning the N-CPPs can cause also the intervening sequences to insert into pSB1C3, I decided to redo some clonings. Since the intervening sequences were designed with unique restriction sites, digestion with these endonucleases should prevent cloning of these.
 +
 +
====Digestion of N-CPP cluster====
 +
[N-CPP plasmid] = 672 ng/μl
 +
 +
''Tested the FastDigest buffer, even though conventional Fermentas restriction enzymes were used.''
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
| 
 +
!width="50"|N-CPP
 +
|-
 +
|colspan="2" align="center"|1st incubation
 +
|-
 +
|10X FastDigest buffer
 +
|align="center"|3
 +
|-
 +
|DNA (2 μg)
 +
|align="center"|3
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|19
 +
|-
 +
|XbaI (conv.)
 +
|align="center"|1
 +
|-
 +
|AgeI (conv.)
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!27 &mu;l
 +
|-
 +
|colspan="2" align="center"|2nd incubation
 +
|-
 +
|FD BamHI
 +
|align="center"|1
 +
|-
 +
|FD HindIII
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!29 &mu;l
 +
|}
 +
 +
*'''1st incubation:''' 37 &deg;C, 2:30
 +
*'''2nd incubation:''' 37 &deg;C, 0:30
 +
*'''Inactivation:''' 80 &deg;C, 20 min
 +
 +
====Ligation====
 +
Two ligation reactions were prepared to test the efficiency of two different ligation buffers.
 +
 +
*'''Vector:''' Dig pSB1C3 X+A EXTR (13.72 ng/&mu;l)
 +
*'''Insert:''' Dig N-CPP X+A 9/9 (31.25 ng/&mu;l)
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|&nbsp;
 +
!Lig pSB1C3<br />NCPP 1<br />9/9
 +
!Lig pSB1C3<br />NCPP 2<br />9/9
 +
|-
 +
|5X Rapid Ligation buf.
 +
|align="center"|4
 +
|align="center"|0
 +
|-
 +
|10X T4 DNA ligase buf.
 +
|align="center"|0
 +
|align="center"|2
 +
|-
 +
|Vector DNA
 +
|align="center"|4
 +
|align="center"|4
 +
|-
 +
|Insert DNA
 +
|align="center"|11
 +
|align="center"|11
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|0
 +
|align="center"|2
 +
|-
 +
|T4 DNA ligase
 +
|align="center"|1
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!20 &mu;l
 +
!20 &mu;l
 +
|}
 +
 +
*Incubation: 22 &deg;C, 16 min
 +
 +
====Digestion of previous ligation sample====
 +
 +
:'''Ligation mix:''' Lig pSB1C3.N-CPP* 6/9
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|Ligation mix
 +
|align="center"|15
 +
|-
 +
|10X FD buffer
 +
|align="center"|2
 +
|-
 +
|FD BamHI
 +
|align="center"|1
 +
|-
 +
|FD HindIII
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!19 &mu;l
 +
|}
 +
 +
*Incubation: 37 &deg;C, 30 min
 +
*Inactivation: 80 &deg;C, 15 min
 +
 +
====Transformations====
 +
Standard transformation protocol.
 +
*3 &mu;l ligation mix
 +
**Lig pSB1C3.N-CPP 1 9/9
 +
**Lig pSB1C3.N-CPP 2 9/9
 +
**Lig pSB1C3.N-CPP * 6/9
 +
*Cm 25 plates
 +
 +
===Joint expression of SOD and yCCS from pEX===
 +
 +
Me and Mimmi were discussing the upcoming expression of SOD and its helper chaperone yCCS. Based on an article by [http://www.ncbi.nlm.nih.gov/pubmed/15358352 Ahl, Lindberg and Tibell (2004)], we decided that the two proteins should be expressed in equal amounts (1:1) from the same vector. Since we only have one expression vector (pEX) available, this requires some modifications.<br />
 +
Our idea is to construct a SOD/yCCS operon from which the two genes can be co-transcribed. This will require a new Shine-Dalgarno (RBS) sequence for translation of the second gene in the operon.
 +
 +
====Extraction of RBS BioBrick (BBa_B0030)====
 +
Extracted BBa_B0030 (RBS 30), carried on pSB1A2, from iGEM plate 1, well 1H. Transformed into Top10.
 +
*Quick transformation
 +
*1 &mu;l DNA
 +
*Amp 100
 +
 +
===Cloning of His&sdot;SOD into pMA===
 +
====Sequencing results====
 +
*pMA.his.SOD_premix ([[media:PMA.his.SOD_premix_9sep.txt|fasta]])
 +
 +
Correct sequence verified by Blastn ([[media:Blastn_pMA.his.SOD_premix-pMA.His*SOD_9sep.txt|results]]). Three silent mutations in the His-tag, as has been previously observed.
 +
 +
 +
 +
 +
 +
 +
 +
== Mimmi ==
 +
 +
=== SOD.his / his.SOD / yCCS ===
 +
 +
==== Plasmid prep. ====
 +
 +
*Follow E.Z.N.A protocol
 +
**Wash 2x with DNA wash buffer
 +
**Eluate in 50µl sH<sub>2</sub>O
 +
 +
{|
 +
! DNA conc.
 +
| ng/µl
 +
|-
 +
| pSB1C3.SOD.his
 +
|
 +
|-
 +
| pSB1C3.his.SOD
 +
|
 +
|-
 +
| pSB1C3.SOD.his
 +
|
 +
|-
 +
 +
 +
==== Glycerol stocks ====
 +
 +
*Add 1800µl to 200µl pre-sterelized glycerol
 +
 +
 +
=== MITF-M ===
 +
 +
==== Site-Directed Mutagenesis ====
 +
 +
{|
 +
! mix
 +
| (µl)
 +
| rowspan="9" width="100" |
 +
! primers
 +
| rowspan="9" width="100" |
 +
! colspan="2" | conditions
 +
| rowspan="3" |
 +
|-
 +
| sH<sub>2</sub>O
 +
| 40
 +
| MITF_1F
 +
! time
 +
! &deg;C
 +
|-
 +
| dNTP
 +
| 1
 +
| MITF_1R
 +
| 2m
 +
| 95
 +
|-
 +
| F primer
 +
| 1
 +
| rowspan="6" |
 +
| 30s
 +
| 95
 +
| )
 +
|-
 +
| R primer
 +
| 1
 +
| 30s
 +
| 55
 +
| > 22 cycles
 +
|-
 +
| Pfu buffer
 +
| 5
 +
| 7m
 +
| 68
 +
| )
 +
|-
 +
| Pfu turbo
 +
| 1
 +
| oo
 +
| 4
 +
| rowspan="3" |
 +
|-
 +
| DNA
 +
| 1
 +
| rowspan="2" |
 +
|-
 +
| align="right" | tot
 +
| 50µl
 +
|}
 +
 +
{{Stockholm/Footer}}

Latest revision as of 11:01, 26 October 2010

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Contents

Andreas

Cloning of N-CPPs into pSB1C3

Since we realized that the method we used for cloning the N-CPPs can cause also the intervening sequences to insert into pSB1C3, I decided to redo some clonings. Since the intervening sequences were designed with unique restriction sites, digestion with these endonucleases should prevent cloning of these.

Digestion of N-CPP cluster

[N-CPP plasmid] = 672 ng/μl

Tested the FastDigest buffer, even though conventional Fermentas restriction enzymes were used.

  N-CPP
1st incubation
10X FastDigest buffer 3
DNA (2 μg) 3
dH2O 19
XbaI (conv.) 1
AgeI (conv.) 1
  27 μl
2nd incubation
FD BamHI 1
FD HindIII 1
  29 μl
  • 1st incubation: 37 °C, 2:30
  • 2nd incubation: 37 °C, 0:30
  • Inactivation: 80 °C, 20 min

Ligation

Two ligation reactions were prepared to test the efficiency of two different ligation buffers.

  • Vector: Dig pSB1C3 X+A EXTR (13.72 ng/μl)
  • Insert: Dig N-CPP X+A 9/9 (31.25 ng/μl)
  Lig pSB1C3
NCPP 1
9/9 		Lig pSB1C3
NCPP 2
9/9
5X Rapid Ligation buf. 4 0
10X T4 DNA ligase buf. 0 2
Vector DNA 4 4
Insert DNA 11 11
dH2O 0 2
T4 DNA ligase 1 1
  20 μl 20 μl
  • Incubation: 22 °C, 16 min

Digestion of previous ligation sample

Ligation mix: Lig pSB1C3.N-CPP* 6/9
Ligation mix 15
10X FD buffer 2
FD BamHI 1
FD HindIII 1
  19 μl
  • Incubation: 37 °C, 30 min
  • Inactivation: 80 °C, 15 min

Transformations

Standard transformation protocol.

  • 3 μl ligation mix
    • Lig pSB1C3.N-CPP 1 9/9
    • Lig pSB1C3.N-CPP 2 9/9
    • Lig pSB1C3.N-CPP * 6/9
  • Cm 25 plates

Joint expression of SOD and yCCS from pEX

Me and Mimmi were discussing the upcoming expression of SOD and its helper chaperone yCCS. Based on an article by [http://www.ncbi.nlm.nih.gov/pubmed/15358352 Ahl, Lindberg and Tibell (2004)], we decided that the two proteins should be expressed in equal amounts (1:1) from the same vector. Since we only have one expression vector (pEX) available, this requires some modifications.
Our idea is to construct a SOD/yCCS operon from which the two genes can be co-transcribed. This will require a new Shine-Dalgarno (RBS) sequence for translation of the second gene in the operon.

Extraction of RBS BioBrick (BBa_B0030)

Extracted BBa_B0030 (RBS 30), carried on pSB1A2, from iGEM plate 1, well 1H. Transformed into Top10.

  • Quick transformation
  • 1 μl DNA
  • Amp 100

Cloning of His⋅SOD into pMA

Sequencing results

  • pMA.his.SOD_premix (fasta)

Correct sequence verified by Blastn (results). Three silent mutations in the His-tag, as has been previously observed.




Mimmi

SOD.his / his.SOD / yCCS

Plasmid prep.

  • Follow E.Z.N.A protocol
    • Wash 2x with DNA wash buffer
    • Eluate in 50µl sH2O

Glycerol stocks

  • Add 1800µl to 200µl pre-sterelized glycerol


MITF-M

Site-Directed Mutagenesis

DNA conc. ng/µl
pSB1C3.SOD.his
pSB1C3.his.SOD
pSB1C3.SOD.his
mix (µl) primers conditions
sH2O 40 MITF_1F time °C
dNTP 1 MITF_1R 2m 95
F primer 1 30s 95 )
R primer 1 30s 55 > 22 cycles
Pfu buffer 5 7m 68 )
Pfu turbo 1 oo 4
DNA 1
tot 50µl





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

Retrieved from "http://2010.igem.org/Team:Stockholm/9_September_2010"