Team:Stockholm/16 September 2010

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{{Stockholm/Top2}}
{{Stockholm/Top2}}
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==Andreas==
==Andreas==
===Assembly of new parts===
===Assembly of new parts===
Line 21: Line 22:
====Colony PCR====
====Colony PCR====
Picked new colonies for colony PCR from 14/9 plates:
Picked new colonies for colony PCR from 14/9 plates:
-
*pSB1K3.TAT⋅SOD⋅His: TAT⋅SH 1-5
+
*pSB1K3.N-TAT⋅SOD⋅His: TAT⋅SH 1-5
-
*pSB1K3.Tra10⋅SOD⋅His: T10⋅SH 1-5
+
*pSB1K3.N-Tra10⋅SOD⋅His: Tra10⋅SH 1-5
*pEX.SOD 1-4
*pEX.SOD 1-4
Line 29: Line 30:
====Gel verification====
====Gel verification====
-
[[image:ColPCR_TAT*SH_16sep.png‎|200px|thumb|right|'''Colony PCR gel verification of N-TAT&sdot;SOD&sdot;His clones.'''<br />4 &mu;l &lambda;; 5 &mu;l sample.<br />1 kb &lambda; = O'GeneRuler 1 kb DNA ladder; 50 bp &lambda; = GeneRuler 50 bp DNA ladder.]]
+
[[image:ColPCR_TAT*SH_16sep.png‎|200px|thumb|right|'''Colony PCR gel verification of N-TAT&sdot;SOD&sdot;His clones. Gel 1'''<br />4 &mu;l &lambda;; 5 &mu;l sample.<br />1 kb &lambda; = O'GeneRuler 1 kb DNA ladder; 50 bp &lambda; = GeneRuler 50 bp DNA ladder.]]
-
[[image:ColPCR_Tra10*SH_16sep.png‎|200px|thumb|right|'''Colony PCR gel verification of N-Tra10&sdot;SOD&sdot;His clones.'''<br />4 &mu;l &lambda;; 5 &mu;l sample.<br />&lambda; = O'GeneRuler 1 kb DNA ladder.]]
+
[[image:ColPCR_Tra10*SH_16sep.png‎|200px|thumb|right|'''Colony PCR gel verification of N-Tra10&sdot;SOD&sdot;His clones. Gel 2'''<br />4 &mu;l &lambda;; 5 &mu;l sample.<br />&lambda; = O'GeneRuler 1 kb DNA ladder.]]
-
[[image:ColPCR_pEX.SOD_16sep.png‎|200px|thumb|right|'''Colony PCR gel verification of pEX.SOD clones.'''<br />4 &mu;l &lambda;; 5 &mu;l sample.<br />&lambda; = O'GeneRuler 1 kb DNA ladder.]]
+
[[image:ColPCR_pEX.SOD_16sep.png‎|200px|thumb|right|'''Colony PCR gel verification of pEX.SOD clones. Gel 3'''<br />4 &mu;l &lambda;; 5 &mu;l sample.<br />&lambda; = O'GeneRuler 1 kb DNA ladder.]]
 +
 
 +
'''Gel 1'''<br />
 +
1 % agarose, 110 V
 +
 
 +
Expected bands
 +
*pSB1K3.N-TAT&sdot;SOD&sdot;His (TAT): 848 bp
 +
*pSB1C3.SOD&sdot;His (pC.SH): 815 bp
 +
 
 +
'''Gel 2'''<br />
 +
1 % agarose, 110 V
 +
 
 +
Expected bands
 +
*pSB1K3.N-Tra10&sdot;SOD&sdot;His (Tra10): 878 bp
 +
*pSB1C3.SOD&sdot;His (pC.SH): 815 bp
 +
 
 +
'''Gel 3'''<br />
 +
1 % agarose, 110 V
 +
 
 +
Expected bands
 +
*pEX.SOD: 678 bp
 +
*pEX.RFP: 862 bp, 1010 bp, 1124 bp, 1272 bp
 +
 
 +
'''Results'''<br />
 +
pSB1K3.N-TAT&sdot;SOD&sdot;His clones 4 and 5 seem slightly larger than the control, pSB1C3.SOD&sdot;His, indicating correct assembly.<br />
 +
Of the pSB1K3.N-Tra10&sdot;SOD&sdot;His samples, clone 5 seems correct, compared to the control (same as above).
 +
For the pEX.SOD samples the results are very strange. Two of the clones resulted in way too large bands (&asymp;2500 bp); unclear what these vectors carry. Clone 2 resulted in a very weak band with the correct size. This clone was chosen for ON growth.
 +
 
 +
====ON cultures====
 +
Set ON cultures (5 ml LB + 50 Km ''or'' 100 Amp; 37 &deg;C, 220 rpm) of the following:
 +
*pSB1K3.N-TAT&sdot;SOD&sdot;His 4
 +
*pSB1K3.N-TAT&sdot;SOD&sdot;His 5
 +
*pSB1K3.N-Tra10&sdot;SOD&sdot;His 5
 +
*pEX.SOD 2
 +
 
 +
===N-CPP sequencing===
 +
Sequencing results from 13/9 returned.
 +
* pSB1C3.nCCP 2 ([[media:PSB1C3.nCCP_2_premix.txt|fasta]])
 +
* pSB1C3.nCCP 3 ([[media:PSB1C3.nCCP_3_premix.txt|fasta]])
 +
* pSB1C3.nCCP 5 ([[media:PSB1C3.nCCP_5_premix.txt|fasta]])
 +
* pSB1C3.nCCP 8 ([[media:PSB1C3.nCCP_8_premix.txt|fasta]])
 +
* pSB1C3.nCCP 9 ([[media:PSB1C3.nCCP_9_premix.txt|fasta]])
 +
* pSB1C3.nCCP 10 ([[media:PSB1C3.nCCP_10_premix.txt|fasta]])
 +
* pSB1C3.nCCP 11 ([[media:PSB1C3.nCCP_11_premix.txt|fasta]])
 +
* pSB1C3.nCCP 12 ([[media:PSB1C3.nCCP_12_premix.txt|fasta]])
 +
 
 +
Ran multiple nucleotide Blast (Blastn) alignments to identify the three N-CPPs from the sequence:
 +
*pSB1C3.N-TAT: clones 9 & 12 ([[media:Blastn_pSB.N-TAT_pSB1C3.nCPP_15sep.txt‎|Blastn]])
 +
*pSB1C3.N-Tra10: clone 5 ([[media:Blastn_pSB.N-Tra10_pSB1C3.nCPP_15sep.txt‎|Blastn]])
 +
*pSB1C3.N-LMWP: clones 2, 3 & 11 ([[media:Blastn_pSB.N-LMWP_pSB1C3.nCPP_15sep.txt‎|Blastn]])
 +
 
 +
====ON cultures====
 +
Set ON cultures for plasmid prep (5 ml LB + 25 Cm; 37 &deg;C, 220 rpm) and glycerol stocks (3 ml LB + 25 Cm; 30 &deg;C).
 +
*Clone 5: pSB1C3.N-Tra10
 +
*Clone 11: pSB1C3.N-LMWP
 +
*Clone 12: pSB1C3.N-TAT
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
== Mimmi ==
 +
 
 +
=== SOD / yCCS ===
 +
 
 +
==== protein gel ====
 +
 
 +
*Thaw the samples and re-heat them in 95&deg;C, 5min
 +
 
 +
*Load them on a PhastGel
 +
 
 +
**20% 8 wells x2
 +
 
 +
{| align="right"
 +
|
 +
! SOD
 +
! yCCS
 +
|-
 +
| length
 +
| 154aa
 +
| 249aa
 +
|-
 +
| Ip
 +
| 6.0695
 +
| 6.6594
 +
|-
 +
| charge
 +
| -2.0
 +
| +0.5
 +
|-
 +
| size
 +
| 15.9kDa
 +
| 27.3kDa
 +
|}
 +
::{|
 +
! SOD.his
 +
| 0h
 +
| 1h
 +
| 2h
 +
| 3h
 +
|-
 +
! his.SOD
 +
| 0h
 +
| 1h
 +
| 2h
 +
| 3h
 +
|-
 +
! yCCS
 +
| 0h
 +
| 1h
 +
| 2h
 +
| 3h
 +
|}
 +
 
 +
 
 +
 
 +
{|
 +
! well
 +
! sample
 +
| rowspan="9" | [[Image:place_for_picture.jpg|200px|thumb|left|]]
 +
| rowspan="9" width="50" |
 +
! well
 +
! sample
 +
| rowspan="9" | [[Image:place_for_picture.jpg|200px|thumb|left|]]
 +
|-
 +
| 1
 +
| ladder
 +
| 1
 +
| ladder
 +
|-
 +
| 2
 +
| SOD.his 0h
 +
| 2
 +
| his.SOD 2h
 +
|-
 +
| 3
 +
| SOD.his 1h
 +
| 3
 +
| his.SOD 3h
 +
|-
 +
| 4
 +
| SOD.his 2h
 +
| 4
 +
| yCCS 0h
 +
|-
 +
| 5
 +
| SOD.his 3h
 +
| 5
 +
| yCCS 1h
 +
|-
 +
| 6
 +
| his.SOD 0h
 +
| 6
 +
| yCCS 2h
 +
|-
 +
| 7
 +
| his.SOD 1h
 +
| 7
 +
| yCCS 3h
 +
|-
 +
| 8
 +
| ladder
 +
| 8
 +
| ladder
 +
|}
 +
 
 +
 
 +
==== glycerol stock / over expression ====
 +
 
 +
''yCCS''
 +
*Pick 2 new colonies, grow ON
 +
 
 +
''SOD.his/his.SOD''
 +
*Pick some cells from the glycerol-stock, grow ON
 +
 
 +
{|
 +
! glycerol stocks
 +
|-
 +
| yCCS 1  3ml LB<sub>AMP</sub> + 2µl culture (from PCR)
 +
|-
 +
| yCCS 2  3ml LB<sub>AMP</sub> + 2µl culture (from PCR)
 +
|-
 +
! ON for expression
 +
|-
 +
| SOD.his 1 5ml LB<sub>AMP</sub> + 5µl culture (from PCR)
 +
|-
 +
| his.SOD 1 5ml LB<sub>AMP</sub> + 5µl culture (from PCR)
 +
|-
 +
| yCCS 1  5ml LB<sub>AMP</sub> + 5µl culture (from PCR)
 +
|-
 +
| yCCS 2  5ml LB<sub>AMP</sub> + 5µl culture (from PCR)
 +
|}
 +
 
 +
 
 +
 
 +
==== PCR ====
 +
 
 +
{|
 +
! mix
 +
| (µl)
 +
| x4
 +
| rowspan="8" width="100" |
 +
! Primers
 +
| rowspan="8" width="100" |
 +
! colspan="2" | conditions
 +
| rowspan="3" |
 +
|-
 +
| mastermix
 +
| 24.5
 +
| rowspan="7" |
 +
| pEX
 +
! time
 +
! &deg;C
 +
|-
 +
| DNA
 +
| 0.5
 +
| rowspan="6" |
 +
| 10m
 +
| 95
 +
|-
 +
| align="right" | tot
 +
| 25µl
 +
| 30s
 +
| 95
 +
| )
 +
|-
 +
| colspan="2" rowspan="4" |
 +
| 30s
 +
| 55
 +
| > 30 cycles
 +
|-
 +
| 1m
 +
| 72
 +
| )
 +
|-
 +
| 5m
 +
| 72
 +
| rowspan="2" |
 +
|-
 +
| oo
 +
| 25
 +
|}
 +
 
 +
 
 +
 
 +
 
 +
=== MITF-M ===
 +
 
 +
==== colony PCR ====
 +
 
 +
{|
 +
! mix
 +
| (µl)
 +
| x8
 +
| rowspan="8" width="100" |
 +
! colspan="2" | conditions
 +
| rowspan="3" |
 +
|-
 +
| mastermix
 +
| 24.5
 +
| rowspan="7" |
 +
! time
 +
! &deg;C
 +
|-
 +
| DNA
 +
| 0.5
 +
| 10m
 +
| 95
 +
|-
 +
| align="right" | tot
 +
| 25µl
 +
| 30s
 +
| 95
 +
| )
 +
|-
 +
| colspan="2" rowspan="4" |
 +
| 30s
 +
| 55
 +
| > 30 cycles
 +
|-
 +
| 2m
 +
| 72
 +
| )
 +
|-
 +
| 10m
 +
| 72
 +
| rowspan="2" |
 +
|-
 +
| oo
 +
| 25
 +
|}
 +
 
 +
 
 +
 
 +
==== Gel ====
 +
 
 +
[[Image:place_for_picture.jpg|200px|thumb|left|]]
 +
{|
 +
! well
 +
! sample
 +
|-
 +
| 1
 +
| ladder
 +
|-
 +
| 2
 +
| MITF-M 1
 +
|-
 +
| 3
 +
| MITF-M 2
 +
|-
 +
| 4
 +
| MITF-M 3
 +
|-
 +
| 5
 +
| MITF-M 4
 +
|-
 +
| 6
 +
| MITF-M 5
 +
|-
 +
| 7
 +
| nTAT
 +
|-
 +
| 8
 +
| cTAT
 +
|-
 +
| 9
 +
| nTra10
 +
|}
 +
 
 +
==Johan==
 +
 
 +
PCR
 +
* 15sep pMA AS bFGF NS
 +
* 15sep pMA EN bFGF EA
 +
 
 +
0,5 µl Pol
 +
 
 +
0,5 µl dNTP
 +
 
 +
5 µl 5x buffer
 +
 
 +
1,5 µl for primer
 +
 
 +
1,5 µl rev primer
 +
 
 +
16 µl H2O
 +
 
 +
3 colonies/plate
 +
 
 +
{{Stockholm/Footer}}

Latest revision as of 01:49, 28 October 2010

Home Project Idea Lab Work Results Modelling Team       Follow us on and        2010.igem.org

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Contents

Andreas

Assembly of new parts

Gel verification of part digestions

Ran gels of digestion samples in parallel with undigested samples to verify successful digestions and insert sizes.

Gel 1

Gel verification of digested samples, gel 1.
&4 μl λ; 3 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.

1 % agarose, 110 V

Gel 2

Gel verification of digested samples, gel 2.
&4 μl λ; 3 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.

1 % agarose, 110 V

Results
Successful digestion with corresponding bands for all digested samples. N-TAT and N-Tra10 not verified, since gel was run too far, but plasmid linearization should indicate successful digestion.

Most samples show somewhat incomplete digestion. For digestions with FastDigest enzymes, this may be an indication of old/inactive enzymes. Especially PstI should be analyzed for activity.

Colony PCR

Picked new colonies for colony PCR from 14/9 plates:

  • pSB1K3.N-TAT⋅SOD⋅His: TAT⋅SH 1-5
  • pSB1K3.N-Tra10⋅SOD⋅His: Tra10⋅SH 1-5
  • pEX.SOD 1-4

Standard colony PCR settings.

  • Elongation: 1:30

Gel verification

Colony PCR gel verification of N-TAT⋅SOD⋅His clones. Gel 1
4 μl λ; 5 μl sample.
1 kb λ = O'GeneRuler 1 kb DNA ladder; 50 bp λ = GeneRuler 50 bp DNA ladder.
Colony PCR gel verification of N-Tra10⋅SOD⋅His clones. Gel 2
4 μl λ; 5 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.
Colony PCR gel verification of pEX.SOD clones. Gel 3
4 μl λ; 5 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.

Gel 1
1 % agarose, 110 V

Expected bands

  • pSB1K3.N-TAT⋅SOD⋅His (TAT): 848 bp
  • pSB1C3.SOD⋅His (pC.SH): 815 bp

Gel 2
1 % agarose, 110 V

Expected bands

  • pSB1K3.N-Tra10⋅SOD⋅His (Tra10): 878 bp
  • pSB1C3.SOD⋅His (pC.SH): 815 bp

Gel 3
1 % agarose, 110 V

Expected bands

  • pEX.SOD: 678 bp
  • pEX.RFP: 862 bp, 1010 bp, 1124 bp, 1272 bp

Results
pSB1K3.N-TAT⋅SOD⋅His clones 4 and 5 seem slightly larger than the control, pSB1C3.SOD⋅His, indicating correct assembly.
Of the pSB1K3.N-Tra10⋅SOD⋅His samples, clone 5 seems correct, compared to the control (same as above). For the pEX.SOD samples the results are very strange. Two of the clones resulted in way too large bands (≈2500 bp); unclear what these vectors carry. Clone 2 resulted in a very weak band with the correct size. This clone was chosen for ON growth.

ON cultures

Set ON cultures (5 ml LB + 50 Km or 100 Amp; 37 °C, 220 rpm) of the following:

  • pSB1K3.N-TAT⋅SOD⋅His 4
  • pSB1K3.N-TAT⋅SOD⋅His 5
  • pSB1K3.N-Tra10⋅SOD⋅His 5
  • pEX.SOD 2

N-CPP sequencing

Sequencing results from 13/9 returned.

Ran multiple nucleotide Blast (Blastn) alignments to identify the three N-CPPs from the sequence:

  • pSB1C3.N-TAT: clones 9 & 12 (Blastn)
  • pSB1C3.N-Tra10: clone 5 (Blastn)
  • pSB1C3.N-LMWP: clones 2, 3 & 11 (Blastn)

ON cultures

Set ON cultures for plasmid prep (5 ml LB + 25 Cm; 37 °C, 220 rpm) and glycerol stocks (3 ml LB + 25 Cm; 30 °C).

  • Clone 5: pSB1C3.N-Tra10
  • Clone 11: pSB1C3.N-LMWP
  • Clone 12: pSB1C3.N-TAT




Mimmi

SOD / yCCS

protein gel

  • Thaw the samples and re-heat them in 95°C, 5min
  • Load them on a PhastGel
    • 20% 8 wells x2
SOD yCCS
length 154aa 249aa
Ip 6.0695 6.6594
charge -2.0 +0.5
size 15.9kDa 27.3kDa
SOD.his 0h 1h 2h 3h
his.SOD 0h 1h 2h 3h
yCCS 0h 1h 2h 3h


well sample
Place for picture.jpg
well sample
Place for picture.jpg
1 ladder 1 ladder
2 SOD.his 0h 2 his.SOD 2h
3 SOD.his 1h 3 his.SOD 3h
4 SOD.his 2h 4 yCCS 0h
5 SOD.his 3h 5 yCCS 1h
6 his.SOD 0h 6 yCCS 2h
7 his.SOD 1h 7 yCCS 3h
8 ladder 8 ladder


glycerol stock / over expression

yCCS

  • Pick 2 new colonies, grow ON

SOD.his/his.SOD

  • Pick some cells from the glycerol-stock, grow ON
glycerol stocks
yCCS 1 3ml LBAMP + 2µl culture (from PCR)
yCCS 2 3ml LBAMP + 2µl culture (from PCR)
ON for expression
SOD.his 1 5ml LBAMP + 5µl culture (from PCR)
his.SOD 1 5ml LBAMP + 5µl culture (from PCR)
yCCS 1 5ml LBAMP + 5µl culture (from PCR)
yCCS 2 5ml LBAMP + 5µl culture (from PCR)


PCR

mix (µl) x4 Primers conditions
mastermix 24.5 pEX time °C
DNA 0.5 10m 95
tot 25µl 30s 95 )
30s 55 > 30 cycles
1m 72 )
5m 72
oo 25



MITF-M

colony PCR

mix (µl) x8 conditions
mastermix 24.5 time °C
DNA 0.5 10m 95
tot 25µl 30s 95 )
30s 55 > 30 cycles
2m 72 )
10m 72
oo 25


Gel

Place for picture.jpg
well sample
1 ladder
2 MITF-M 1
3 MITF-M 2
4 MITF-M 3
5 MITF-M 4
6 MITF-M 5
7 nTAT
8 cTAT
9 nTra10

Johan

PCR

  • 15sep pMA AS bFGF NS
  • 15sep pMA EN bFGF EA

0,5 µl Pol

0,5 µl dNTP

5 µl 5x buffer

1,5 µl for primer

1,5 µl rev primer

16 µl H2O

3 colonies/plate





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

Retrieved from "http://2010.igem.org/Team:Stockholm/16_September_2010"