Team:Stockholm/13 August 2010

From 2010.igem.org

(Difference between revisions)
(Nina)
(Andreas)
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==Andreas==
==Andreas==
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===Site-directed mutagenesis of SOD & yCCS===
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====Transformation results====
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*SOD-->Amp 100: Few colonies, probably due to contaminated competent cells.
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*SOD-->Cm 25: Good colony yield
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*yCCS-->Cm 25: Good colony yield
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====Colony PCR====
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*SOD-->Amp: 4 colonies (SA1-SA4)
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*SOD-->Cm: 5 colonies (SC1-SC5)
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*yCCS-->Cm: 5 colonies (yC1-yC5)
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*Positive control: pSB1C3.BBa_J18932 plasmid (PC)
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Procedures as described in the colony PCR protocol. '''Elongation time:''' 1:40
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====yCCS digestion====
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Amplified yCCS DNA was digested to verify successful site-directed mutagenesis (i.e. EcoRI site removal).
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*2 μl 10X FD Green buffer
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*17 μl DNA
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*1 μl FastDigest EcoRI
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Incubation: 37 °C, 10 min
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====Gel verification====
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[[image:ColPCR_SOD-yCCS_13aug.png|200px|thumb|Colony PCR verification of mutagenized yCCS and SOD.<br>1 % agarose, 110 V, 40 min.<br>'''Row 1:'''1 kb &lambda;, SA1, SA2, SA3, SA4, SC1, SC2, SC3, SC4, SC5, d.yC5<br>
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'''Row 2:''' 1 kb &lambda;, yC1, d.yC1, yC2, d.yC2, yC3, d.yC3, yC4, d.yC4, yC5, PC<br>&lambda;: DNA ladder. d.:digested sample.]]
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1 % agarose, 110 V, 40 min
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=====Results=====
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Due to very unclear and weak bands, I decided to discard the gel and run a new on Monday.

Revision as of 15:02, 16 August 2010


Contents

Nina


Gel verification of protein A (ZZ domain) and IgG protease

I ran a new agarose gel 1 % on my protein A and IgG protease samples. However I didn't load the positive protein A control sample since I realized that it would not fullfill its purpose considering the vector verification primer I used does not bind to the vector protein A sits in, which is the original vector I obtained it in.

Ladder: GeneRuler™ DNA Ladder Mix, ready-to-use, 100-10,000 bp Fermentas

Arrangement on gels:

Gel1.jpg Prot.jpg


Colony-PCR of site-directed mutagenesis Tyrosinase

Colony nr: 2, 4, 6 & 8.

PCR Mix:

Total volume 100 ul, aliquate 25 ul in the four pcr tubes.

  • F primer 50 uM 1 ul
  • R primer 50 uM 1 ul
  • dNTP 10 uM 1 ul
  • Buffer phusion 5X 20 ul
  • Phusion polymerase 1 ul
  • H2O 75 ul

PCR prgm:

98 °C 2 min

22 cycles of:

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

72 °C 1 min

4 °C ∞

I ran 5 ul of the PCR products with 1 ul loading dye 6X on an agarose gel 1 % 80 V.

Ladder: GeneRuler™ DNA Ladder Mix, ready-to-use, 100-10,000 bp Fermentas

Arrangement on gels:

Tyr-site.jpg

Tyr.jpg


Overnight culture of protein A (ZZ domain) and Tyrosinase

  • I inoculated protein A ZZ domain inserted in the bank vector C (with chloramphenicol) colony # 5 in 12 ml LB and 24 ul chloramphenicol 50 mg/ml. This was incubated O/N in 37 °C with shake.
  • I inoculated tyrosinase in its original vector colonies # 2, 4, 6 & 8 in 12 ml LB and 24 ul ampicillin 50 mg/ml. This was incubated O/N in 37 °C with shake.

Sequencing of protein A (ZZ domain)

I prepared a tube of protein A ZZ domain for sequencing:

15 ul plasmid from a mini-prep and 1.5 ul (10uM) primer. Preferably the forward primer (VF) of the vectors verification primers.

  • ASB0045 303

Andreas

Site-directed mutagenesis of SOD & yCCS

Transformation results

  • SOD-->Amp 100: Few colonies, probably due to contaminated competent cells.
  • SOD-->Cm 25: Good colony yield
  • yCCS-->Cm 25: Good colony yield

Colony PCR

  • SOD-->Amp: 4 colonies (SA1-SA4)
  • SOD-->Cm: 5 colonies (SC1-SC5)
  • yCCS-->Cm: 5 colonies (yC1-yC5)
  • Positive control: pSB1C3.BBa_J18932 plasmid (PC)

Procedures as described in the colony PCR protocol. Elongation time: 1:40

yCCS digestion

Amplified yCCS DNA was digested to verify successful site-directed mutagenesis (i.e. EcoRI site removal).

  • 2 μl 10X FD Green buffer
  • 17 μl DNA
  • 1 μl FastDigest EcoRI

Incubation: 37 °C, 10 min

Gel verification

Colony PCR verification of mutagenized yCCS and SOD.
1 % agarose, 110 V, 40 min.
Row 1:1 kb λ, SA1, SA2, SA3, SA4, SC1, SC2, SC3, SC4, SC5, d.yC5
Row 2: 1 kb λ, yC1, d.yC1, yC2, d.yC2, yC3, d.yC3, yC4, d.yC4, yC5, PC
λ: DNA ladder. d.:digested sample.

1 % agarose, 110 V, 40 min

Results

Due to very unclear and weak bands, I decided to discard the gel and run a new on Monday.