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°C, T<sub>a</sub>2 = 62.6°C | ||
+ | |||
+ | |||
+ | ::'''Primers''' | ||
+ | :(MW/10)/(OD*33)=df | ||
+ | ::1000/df=V for 100µM | ||
+ | :::1:10=V for 10µM | ||
+ | |||
+ | |||
+ | :*MITF_F: 70.59µl H<sub>2</sub>O --> 1:10 | ||
+ | :*MITF_R: 541.88µl H<sub>2</sub>O --> 1:10 | ||
+ | |||
+ | |||
+ | |||
+ | {| cellspacing="0" | ||
+ | ! Mix | ||
+ | | (µl) | ||
+ | | X4 (µl) | ||
+ | | rowspan="11" width="150" | | ||
+ | ! Mix control | ||
+ | | (µl) | ||
+ | | X5 (µl) | ||
+ | | rowspan="11" width="150" | | ||
+ | ! colspan="2" | contitions | ||
+ | | rowspan="3" width="150" | | ||
+ | |- | ||
+ | | H<sub>2</sub>O | ||
+ | | 39.5 | ||
+ | | 158 | ||
+ | | H<sub>2</sub>O | ||
+ | | 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 | ||
+ | | align="right" | tot | ||
+ | | 25 | ||
+ | | 125 | ||
+ | | 1m30s | ||
+ | | 68 | ||
+ | | ) | ||
+ | |- | ||
+ | | dNTPs | ||
+ | | 1.5 | ||
+ | | 6 | ||
+ | | rowspan="6" colspan="3" | | ||
+ | | 30s | ||
+ | | 94 | ||
+ | | ) | ||
+ | |- | ||
+ | | MgSO<sub>4</sub> | ||
+ | | 1 | ||
+ | | 4 | ||
+ | | 30s | ||
+ | | 62 | ||
+ | | > 25 cycles | ||
+ | |- | ||
+ | | DNA | ||
+ | | 1 | ||
+ | | 4 | ||
+ | | 1m30s | ||
+ | | 68 | ||
+ | | ) | ||
+ | |- | ||
+ | | align="right" | tot | ||
+ | | 50 | ||
+ | | 200 | ||
+ | | 10m | ||
+ | | 68 | ||
+ | | rowspan="2" | | ||
+ | |- | ||
+ | | colspan="3"| | ||
+ | | oo | ||
+ | | 10 | ||
+ | |} |
Revision as of 14:13, 9 August 2010
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
- 1000/df=V for 100µM
- 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 |