Team:Stockholm/19 August 2010

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(Difference between revisions)
(→Andreas)
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SOD 1 sample and glycerol stock will be discarded; the other clones are saved as verified.
SOD 1 sample and glycerol stock will be discarded; the other clones are saved as verified.
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== Mimmi ==
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=== SOD / yCCS ===
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==== Digestion ====
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{|
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| colspan="3" | '''''pSB1C3.SOD/pSB1C3.yCCS'''''
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| width="150" rowspan="11" |
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| colspan="2" | '''''pMA.His'''''
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| width="150" rowspan="11" |
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| colspan="2" | '''''pSB1K3.RFP'''''
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| width="150" rowspan="11" |
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|-
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| colspan="3" |
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| colspan="2" |
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| colspan="2" |
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|-
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| colspan="3" | 8< &nbsp; EcoRI + AgeI
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| colspan="2" | 8< &nbsp; NgoMIV + PstI
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| colspan="2" | 8< &nbsp; EcoRI + PstI
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|-
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| colspan="3" |
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| colspan="2" |
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| colspan="2" |
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|-
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! Mix
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| SOD
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| yCCS
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| rowspan="4" |
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| His-tag
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| rowspan="4" |
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| RFP
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|-
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| sH<sub>2</sub>O
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| 7
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| 6
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| 14
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| 11
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|-
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| DNA
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| 19
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| 20
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| 12
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| 15
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|-
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| 10X FD buffer
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| 3
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| 3
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| 3
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| 3
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|-
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| EcoRI
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| 0.5
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| 0.5
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| NgoMIV
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| 0.5
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| EcoRI
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| 0.5
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|-
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| AgeI
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| 0.5
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| 0.5
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| PstI
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| 0.5
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| PstI
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| 0.5
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|-
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| align="right" | tot
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| 30µl
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| 30µl
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| align="right" | tot
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| 30µl
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| align="right" | tot
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| 30µl
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|}
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:*Incubate with just NgoMIV in 37&deg;C for 1h 30m
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:*Add the other restriction enzymes and incubate in 37&deg;C for 30m
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:*Inactivate restriction enzymes in 80&deg;C for 5m

Revision as of 11:11, 20 August 2010

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Contents

Andreas

Site-directed mutagenesis of SOD & yCCS

Sequencing results

Received the sequencing results for m-SOD 1, m-SOD 2, m-yCCS 1 and m-yCCS 4 from Eurofins. Ran a nucleotide BLAST (BLASTn) alignment to verify successful site-directed mutagenesis.

All sequencings revealed an insertion in the Freiburg suffix, between SpeI and PstI restriction sites. We were already aware of this, and this will be taken care of later by digestion and cloning with SpeI, thereby removing the insertion.
BLASTn results also revealed three mutations in both yCCS sequences. A protein BLAST (BLASTp) alignment of the translated yCCS sequences revealed all four mutations as silent mutations (BLASTp results).

SOD 1 sample and glycerol stock will be discarded; the other clones are saved as verified.


Mimmi

SOD / yCCS

Digestion

pSB1C3.SOD/pSB1C3.yCCS pMA.His pSB1K3.RFP
8<   EcoRI + AgeI 8<   NgoMIV + PstI 8<   EcoRI + PstI
Mix SOD yCCS His-tag RFP
sH2O 7 6 14 11
DNA 19 20 12 15
10X FD buffer 3 3 3 3
EcoRI 0.5 0.5 NgoMIV 0.5 EcoRI 0.5
AgeI 0.5 0.5 PstI 0.5 PstI 0.5
tot 30µl 30µl tot 30µl tot 30µl


  • Incubate with just NgoMIV in 37°C for 1h 30m
  • Add the other restriction enzymes and incubate in 37°C for 30m
  • Inactivate restriction enzymes in 80°C for 5m
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