Team:Stockholm/5 July 2010

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__TOC__
__TOC__
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= Hassan =
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= Andreas =
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=== Plasmid preparations ===
 +
Prepared plasmids from ON cultures.
 +
 
 +
'''DNA concentrations'''
 +
{|border="1" cellspacing="0" cellpadding="2"
 +
|'''Sample'''
 +
|'''DNA conc (ng/ul)'''
 +
|'''A<sub>260</sub>/A<sub>280</sub>'''
 +
|-
 +
|pSB1A3.BBa_J04450
 +
|66.8
 +
|1.74
 +
|-
 +
|pSB1C3.BBa_J04450
 +
|36.5
 +
|1.87
 +
|-
 +
|pSB1AC3.BBa_J04450
 +
|41.0
 +
|1.95
 +
|-
 +
|pSB1AK3.BBa_J04450
 +
|10.8
 +
|2.64
 +
|-
 +
|pMA.BBa_J18930
 +
|33.3
 +
|1.95
 +
|-
 +
|pMA.BBa_J18931
 +
|52.5
 +
|1.92
 +
|-
 +
|pMA.BBa_J18932
 +
|34.1
 +
|1.99
 +
|}
 +
 
 +
=== Part and primer design ===
 +
 
 +
I investigated Mimmi's primers, most specifically the ones designed for ligation of our CPPs (TAT, LMWP and Transportan 10). Me and Johan discussed whether we should order them as is, or if we should instead have the genes synthesized. We decided that we should rethink the matter until tomorrow, when we can have a group meeting and discuss this further.
 +
 
 +
Johan also discovered that several of our genes contain disallowed BioBrick restriction sites:
 +
* '''Tyrosinase''' contains one NgoMIV site at 500 bp.
 +
* '''yCCS''' contains one EcoRI site at 230 bp.
 +
* '''bFGF''' contains one AgeI site at 350 bp.
 +
 
 +
These need to be removed. We will discuss this tomorrow.
 +
 
 +
=Johan=
 +
 
 +
* PCR bFGF, 16 colonies
 +
 
 +
* gel
 +
 
 +
All bands too small (250) nt. After discussion with Rob we looked at the sequence and found that the gene has a AgeI restriction site. '''NOT GOOD'''
 +
 
 +
---
 +
 
 +
* Transformation
 +
 
 +
bFGF vector (Amp resistance) from Japan into competent BL21 cells
 +
 
 +
50 µl spreaded on one plate, 50 µl 10x dilution on one plate
 +
 
 +
{{Stockholm/Footer}}

Latest revision as of 18:42, 27 October 2010

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Contents


Andreas

Plasmid preparations

Prepared plasmids from ON cultures.

DNA concentrations

Sample DNA conc (ng/ul) A260/A280
pSB1A3.BBa_J04450 66.8 1.74
pSB1C3.BBa_J04450 36.5 1.87
pSB1AC3.BBa_J04450 41.0 1.95
pSB1AK3.BBa_J04450 10.8 2.64
pMA.BBa_J18930 33.3 1.95
pMA.BBa_J18931 52.5 1.92
pMA.BBa_J18932 34.1 1.99

Part and primer design

I investigated Mimmi's primers, most specifically the ones designed for ligation of our CPPs (TAT, LMWP and Transportan 10). Me and Johan discussed whether we should order them as is, or if we should instead have the genes synthesized. We decided that we should rethink the matter until tomorrow, when we can have a group meeting and discuss this further.

Johan also discovered that several of our genes contain disallowed BioBrick restriction sites:

  • Tyrosinase contains one NgoMIV site at 500 bp.
  • yCCS contains one EcoRI site at 230 bp.
  • bFGF contains one AgeI site at 350 bp.

These need to be removed. We will discuss this tomorrow.

Johan

  • PCR bFGF, 16 colonies
  • gel

All bands too small (250) nt. After discussion with Rob we looked at the sequence and found that the gene has a AgeI restriction site. NOT GOOD

---

  • Transformation

bFGF vector (Amp resistance) from Japan into competent BL21 cells

50 µl spreaded on one plate, 50 µl 10x dilution on one plate





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

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