Team:Stockholm/2 August 2010

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Andreas

CPP DNA synthesis

Designed a CPP cluster to be sent for synthesis, containing all CPPs (with Freiburg prefixes and suffixes) as follows:

(ApaI)—C-Tra10—(BamHI)—N-Tra10—(ClaI)—C-TAT—(NcoI)—N-TAT—(SmaI)—C-LMWP—(BglII)—N-LMWP—(HindIII)

>CPP_cluster
GGGCCCGCGAATTCGCGGCCGCTTCTAGATGGCCGGCGCGGGTTACCTGCTGGGTAAAAT
CAACCTGAAAGCGCTGGCGGCGCTGGCGAAAAAAATCCTGACCGGTTAATACTAGTAGCG
GCCGCTGCAGGCGGATCCGCGAATTCGCGGCCGCTTCTAGATGGCGGGTTACCTGCTGGG
TAAAATCAACCTGAAAGCGCTGGCGGCGCTGGCGAAAAAAATCCTGACCGGTTAATACTA
GTAGCGGCCGCTGCAGGCATCGATGCGAATTCGCGGCCGCTTCTAGATGGCCGGCTACGG
TCGTAAAAAACGTCGTCAGCGTCGTCGTACCGGTTAATACTAGTAGCGGCCGCTGCAGGC
CCATGGGCGAATTCGCGGCCGCTTCTAGATGTACGGTCGTAAAAAACGTCGTCAGCGTCG
TCGTACCGGTTAATACTAGTAGCGGCCGCTGCAGGCCCCGGGGCGAATTCGCGGCCGCTT
CTAGATGGCCGGCGTTTCTCGTCGTCGTCGTCGTCGTGGTGGTCGTCGTCGTCGTACCGG
TTAATACTAGTAGCGGCCGCTGCAGGCAGATCTGCGAATTCGCGGCCGCTTCTAGATGGT
TTCTCGTCGTCGTCGTCGTCGTGGTGGTCGTCGTCGTCGTACCGGTTAATACTAGTAGCG
GCCGCTGCAGGCAAGCTT

Cm stocks

Prepared 4 x 1 ml 25 μg/μl Cm stocks.

LB agar plates

Prepared Cm and Amp LB agar plates as follows:

  • 20x 25 μg/ml Cm
  • 20x 100 μg/ml Amp

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Mimmi

MITF - amplifying and moving the gene to pSB1C3

  • Using AmplifX, suggested Ta1 = 54.8°C, Ta2 = 62.6°C


Primers
(MW/10)/(OD*33)=df
1000/df=V for 100µM
1:10=V for 10µM


  • MITF_F: 70.59µl H2O --> 1:10
  • MITF_R: 541.88µl H2O --> 1:10


Mix (µl) X4 (µl) Mix control (µl) X5 (µl) contitions
H2O 39.5 158 H2O 22.5 112.5 time °C
F primer 0.75 3 F primer 1 5 2m 94
R primer 0.75 3 R primer 1 5 30s 94 )
buffer 5 20 DNA 0.5 5*0.5 30s 55 > 5 cycles
Pfx pol 0.5 2 tot 25 125 1m30s 68 )
dNTPs 1.5 6 30s 94 )
MgSO4 1 4 30s 62 > 25 cycles
DNA 1 4 1m30s 68 )
tot 50 200 10m 68
oo 10

Nina


PCR on Tyrosinase

I ran a new PCR on the Tyrosinase since I realized that the band from the last PCR on this gene was incorrect (too high than expected). The gene Tyrosinase is about 1900 nt.

PCR Mix (100 ul):

  • Fprimer 50 uM 1 ul
  • Rprimer 50 uM 1 ul
  • dNTP 10 mM 2 ul
  • Buffer 5X 20 ul for phusion polymerase
  • Buffer 10X 10 ul for fermenta (Pfu) polymerase
  • phusion polymerase 1 ul
  • fermenta (Pfu) polymerase 1 ul
  • H2O 75 ul for phusion polymerase
  • H2O 85 ul for fermenta (Pfu) polymerase
  • DNA template 1 colony

Primer dilutions:

100 uM gets a 1:2 dilution to become 50 uM

cV = cV

100V = 50*10ul

V = 500 / 100 = 5 ul primer and fill up to 10 ul with H2O.

I aliquate the 100 ul in four PCR tubes, each with 25 ul PCR mix.

PCR prgm:

98 °C 2 min

2 * 29 cycles of:

  • 98 °C 30 sec
  • 50-72 °C 30 sec
  • 72 °C 2 min

72 °C 1 min

4 °C ∞


Agarose gel on Tyrosinase

I ran a 1 % agarose gel on the tyrosinase PCR productes.

Ladder: FastRuler™ Middle Range DNA Ladder, ready-to-use, 100-5000 bp Fermentas

Arrangement on gels:

Steg.jpg

1 = 50 °C - 4 = 72 °C

Tyrsuc.jpg

P1 and 2 look like they have succesfully PCR:ed tyrosinase.


Sequencing yCCS

I send two samples of yCCS for sequencing.

  • 15 ul vector
  • 1.5 ul 10uM VF2 primer

A: ASB0045 246

B: ASB0045 245





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