Team:Stockholm/14 September 2010

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(New page: {{Stockholm/Top2}} ==Andreas==)
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{{Stockholm/Top2}}
{{Stockholm/Top2}}
==Andreas==
==Andreas==
 +
===Preparation of Top10 chemically competent cells===
 +
No growth on neither Amp 100, Cm 25 nor Km 50 plates - no contamination of competent cells.
 +
 +
===pSB1C3.N-TAT & .N-Tra10===
 +
Successful transformation. One colony of each plate picked and dissolved in 10 μl and saved in fridge for later glycerol stock preparation.
 +
 +
===Assembly of new parts===
 +
Planned and designed cloning strategies for some of the assemblies I will be constructing:
 +
*pEX.SOD
 +
*N-TAT⋅SOD⋅His
 +
*N-Tra10⋅SOD⋅His
 +
*N-LMWP&sdot;SOD&sdot;His (<span style="color:red">N-LMWP n/a</span>)
 +
*His&sdot;SOD&sdot;C-Tra10 (<span style="color:red">C-Tra10 n/a</span>)
 +
*His&sdot;SOD&sdot;C-TAT (<span style="color:red">C-TAT n/a</span>)
 +
*His&sdot;SOD&sdot;C-LMWP (<span style="color:red">C-LMWP n/a</span>)
 +
*RBS.yCCS
 +
 +
====Digestions====
 +
*[pSB1C3.SOD] = 105.5 ng/&mu;l
 +
*[pSB1A2.RBS 34] = 60 ng/&mu;l
 +
*[pMA.His&sdot;SOD] = 266 ng/&mu;l
 +
*[pMA.SOD&sdot;His] = 246 ng/&mu;l
 +
*[pSB1C3.N-TAT] = 200 ng/&mu;l
 +
*[pSB1C3.N-Tra10] = 151 ng/&mu;l
 +
*[pSB1K3.RFP] = 104 ng/&mu;l
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|&nbsp;
 +
!width="50"|His&sdot;SOD
 +
!width="50"|SOD&sdot;His
 +
!width="50"|RBS 34
 +
!width="50"|pSB1K3
 +
!width="50"|N-TAT
 +
!width="50"|N-Tra10
 +
!width="50"|SOD
 +
|-
 +
|10X FD buffer
 +
|align="center"|3
 +
|align="center"|3
 +
|align="center"|3
 +
|align="center"|3
 +
|align="center"|3
 +
|align="center"|3
 +
|align="center"|1
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|16.9
 +
|align="center"|17.5
 +
|align="center"|0
 +
|align="center"|5.8
 +
|align="center"|15
 +
|align="center"|11.8
 +
|align="center"|7
 +
|-
 +
|DNA
 +
|align="center"|8.1
 +
|align="center"|7.5
 +
|align="center"|25
 +
|align="center"|19.2
 +
|align="center"|10
 +
|align="center"|13.2
 +
|align="center"|1
 +
|-
 +
|NgoMIV (NEB)
 +
|align="center"|1
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|0
 +
|-
 +
|PstI
 +
|align="center"|1
 +
|align="center"|0
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|1
 +
|-
 +
|FD SpeI
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|1
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|0
 +
|-
 +
|FD EcoRI
 +
|align="center"|0
 +
|align="center"|1
 +
|align="center"|0
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|0
 +
|-
 +
|AgeI
 +
|align="center"|0
 +
|align="center"|1
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|0
 +
|-
 +
|FD XbaI
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!30 &mu;l
 +
!30 &mu;l
 +
!30 &mu;l
 +
!30 &mu;l
 +
!30 &mu;l
 +
!30 &mu;l
 +
!10 &mu;l
 +
|}
 +
 +
*Incubation: 37 &deg;C
 +
**2:00 (conventional enzymes)
 +
**0:30 (FastDigest enzymes)
 +
*Inactivation: 80 &deg;C, 20 min
 +
 +
====Ligations====
 +
[Dig RBS 34 14/9] = 50 ng/&mu;l
 +
[Dig pSB1K3 14/9] = 66.6 ng/&mu;l
 +
[Dig SOD&sdot;His 14/9] = 66.6 ng/&mu;l
 +
[Dig His&sdot;SOD 14/9] = 66.6 ng/&mu;l
 +
[Dig N-TAT 14/9] = 66.6 ng/&mu;l
 +
[Dig N-Tra10 14/9] = 66.6 ng/&mu;l
 +
[Dig SOD 14/9] = 15 ng/&mu;l
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|&nbsp;
 +
!width="50"|Lig pEX.SOD 14/9
 +
!width="50"|Lig pK.N-TAT&sdot; SOD&sdot;His 14/9
 +
!width="50"|Lig pK.N-Tra10&sdot; SOD&sdot;His 14/9
 +
!width="50"|Lig pA.RBS. yCCS 14/9
 +
|-
 +
|10X T4 ligase buffer
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|-
 +
|Vector DNA
 +
|align="center"|3
 +
|align="center"|1.5
 +
|align="center"|1.5
 +
|align="center"|2
 +
|-
 +
|Insert DNA (1)
 +
|align="center"|10
 +
|align="center"|11
 +
|align="center"|11
 +
|align="center"|6
 +
|-
 +
|Insert DNA (2)
 +
|align="center"|&ndash;
 +
|align="center"|4.5
 +
|align="center"|4.5
 +
|align="center"|&ndash;
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|4
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|9
 +
|-
 +
|T4 DNA ligase
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!20 &mu;l
 +
!20 &mu;l
 +
!20 &mu;l
 +
!20 &mu;l
 +
|}
 +
 +
*Incubation: 22 &deg;C, 15 min
 +
 +
====Transformation====
 +
Standard transformation
 +
*1 &mu;l ligation mix
 +
*15 min on ice
 +
*50 &mu;l IPTG (on pEX.SOD Amp 100 plate only)

Revision as of 19:38, 14 September 2010

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Contents

Andreas

Preparation of Top10 chemically competent cells

No growth on neither Amp 100, Cm 25 nor Km 50 plates - no contamination of competent cells.

pSB1C3.N-TAT & .N-Tra10

Successful transformation. One colony of each plate picked and dissolved in 10 μl and saved in fridge for later glycerol stock preparation.

Assembly of new parts

Planned and designed cloning strategies for some of the assemblies I will be constructing:

  • pEX.SOD
  • N-TAT⋅SOD⋅His
  • N-Tra10⋅SOD⋅His
  • N-LMWP⋅SOD⋅His (N-LMWP n/a)
  • His⋅SOD⋅C-Tra10 (C-Tra10 n/a)
  • His⋅SOD⋅C-TAT (C-TAT n/a)
  • His⋅SOD⋅C-LMWP (C-LMWP n/a)
  • RBS.yCCS

Digestions

  • [pSB1C3.SOD] = 105.5 ng/μl
  • [pSB1A2.RBS 34] = 60 ng/μl
  • [pMA.His⋅SOD] = 266 ng/μl
  • [pMA.SOD⋅His] = 246 ng/μl
  • [pSB1C3.N-TAT] = 200 ng/μl
  • [pSB1C3.N-Tra10] = 151 ng/μl
  • [pSB1K3.RFP] = 104 ng/μl
  His⋅SOD SOD⋅His RBS 34 pSB1K3 N-TAT N-Tra10 SOD
10X FD buffer 3 3 3 3 3 3 1
dH2O 16.9 17.5 0 5.8 15 11.8 7
DNA 8.1 7.5 25 19.2 10 13.2 1
NgoMIV (NEB) 1 0 0 0 0 0 0
PstI 1 0 1 1 0 0 1
FD SpeI 0 0 1 0 0 0 0
FD EcoRI 0 1 0 1 1 1 0
AgeI 0 1 0 0 1 1 0
FD XbaI 0 0 0 0 0 0 1
  30 μl 30 μl 30 μl 30 μl 30 μl 30 μl 10 μl
  • Incubation: 37 °C
    • 2:00 (conventional enzymes)
    • 0:30 (FastDigest enzymes)
  • Inactivation: 80 °C, 20 min

Ligations

[Dig RBS 34 14/9] = 50 ng/μl [Dig pSB1K3 14/9] = 66.6 ng/μl [Dig SOD⋅His 14/9] = 66.6 ng/μl [Dig His⋅SOD 14/9] = 66.6 ng/μl [Dig N-TAT 14/9] = 66.6 ng/μl [Dig N-Tra10 14/9] = 66.6 ng/μl [Dig SOD 14/9] = 15 ng/μl

  Lig pEX.SOD 14/9 Lig pK.N-TAT⋅ SOD⋅His 14/9 Lig pK.N-Tra10⋅ SOD⋅His 14/9 Lig pA.RBS. yCCS 14/9
10X T4 ligase buffer 2 2 2 2
Vector DNA 3 1.5 1.5 2
Insert DNA (1) 10 11 11 6
Insert DNA (2) – 4.5 4.5 –
dH2O 4 0 0 9
T4 DNA ligase 1 1 1 1
  20 μl 20 μl 20 μl 20 μl
  • Incubation: 22 °C, 15 min

Transformation

Standard transformation

  • 1 μl ligation mix
  • 15 min on ice
  • 50 μl IPTG (on pEX.SOD Amp 100 plate only)
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