Team:Stockholm/2 August 2010

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(Difference between revisions)
(New page: {{Stockholm/Top2}} ==Andreas== ===CPP DNA synthesis=== Designed a CPP cluster to be sent for synthesis, containing all CPPs (with Freiburg prefixes and suffixes) as follows: (ApaI)—''...)
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*20x 25 μg/ml Cm
*20x 25 μg/ml Cm
*20x 100 μg/ml Amp
*20x 100 μg/ml Amp
 +
 +
---
 +
 +
== Mimmi ==
 +
 +
 +
=== MITF - amplifying and moving the gene to pSB1C3 ===
 +
 +
 +
*Using AmplifX, suggested T<sub>a</sub>1 = 54.8&deg;C, T<sub>a</sub>2 = 62.6&deg;C
 +
 +
 +
::'''Primers'''
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:(MW/10)/(OD*33)=df
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::1000/df=V for 100µM
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:::1:10=V for 10µM
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 +
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:*MITF_F: 70.59µl H<sub>2</sub>O --> 1:10
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:*MITF_R: 541.88µl H<sub>2</sub>O --> 1:10
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 +
 +
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{| cellspacing="0"
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! Mix
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| (µl)
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| X4 (µl)
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| rowspan="11" width="150" |
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! Mix control
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| (µl)
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| X5 (µl)
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| rowspan="11" width="150" |
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! colspan="2" | contitions
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| rowspan="3" width="150" |
 +
|-
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| H<sub>2</sub>O
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| 39.5
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| 158
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| H<sub>2</sub>O
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| 22.5
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| 112.5
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! time
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! &deg;C
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|-
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| F primer
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| 0.75
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| 3
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| F primer
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| 1
 +
| 5
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| 2m
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| 94
 +
|-
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| R primer
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| 0.75
 +
| 3
 +
| R primer
 +
| 1
 +
| 5
 +
| 30s
 +
| 94
 +
| )
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|-
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| buffer
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| 5
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| 20
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| DNA
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| 0.5
 +
| 5*0.5
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| 30s
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| 55
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| > 5 cycles
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|-
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| Pfx pol
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| 0.5
 +
| 2
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| align="right" | tot
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| 25
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| 125
 +
| 1m30s
 +
| 68
 +
| )
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|-
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| dNTPs
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| 1.5
 +
| 6
 +
| rowspan="6" colspan="3" |
 +
| 30s
 +
| 94
 +
| )
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|-
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| MgSO<sub>4</sub>
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| 1
 +
| 4
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| 30s
 +
| 62
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| > 25 cycles
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|-
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| DNA
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| 1
 +
| 4
 +
| 1m30s
 +
| 68
 +
| )
 +
|-
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| align="right" | tot
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| 50
 +
| 200
 +
| 10m
 +
| 68
 +
| rowspan="2" |
 +
|-
 +
| colspan="3"|
 +
| oo
 +
| 10
 +
|}

Revision as of 14:13, 9 August 2010

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Contents

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

---

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
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