Team:Stockholm/21 August 2010

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Hassan

found some extra articles for gene-regulatory networks: [http://www.ncbi.nlm.nih.gov/pubmed/17504165] and [http://www.biomedcentral.com/1471-2105/8/S6/S9]

Andreas

Assembly of CPP⋅protein⋅His constructs

Assembly of His⋅SOD/yCCS and SOD/yCCS⋅His into pSB1K3

Step I in cloning strategy (19/8)

Transformation results
  • pSB1K3.SOD⋅His
  • pSB1K3.yCCS⋅His
  • pSB1K3.His⋅SOD
  • pSB1K3.His⋅yCCS

Good yield on all plates.

Colony PCR

Three white colonies picked from each plate for verification.

  • pSB1K3.SOD⋅His: 1=A1, 2=A2, 3=A3
  • pSB1K3.yCCS⋅His: 1=A4, 2=A5, 3=A6
  • pSB1K3.His⋅SOD: 1=A7, 2=A8, 3=A9
  • pSB1K3.His⋅yCCS: 1=A10, 2=A11, 3=A12
  • Blank: A13

Procedures as described in colony PCR protocol.

  • Forward primer: VF2
  • Reverse primer: VR
  • Elongation time: 2:00
Gel verification
Gel verification of pEX.RFP constructs and pEX vector.
Loading: λ, A1, A8, A2, A3, A4, A5, A6, A7, λ
3 μl λ, 4 μl sample. λ = O'GeneRuler 1 kb DNA ladder.

1 % agarose, 90 V, 1 h.

Transfer of RFP to pEX

Continued from 19/8

Colony PCR

Since the gel results from 20/8 only verified pMA.His, a new colony PCR was run to verify pEX and pEX.RFP. Two new clones (5 & 6) of pEX.RFP were picked and run with clones 2 and 3 from 20/8. To test whether the red colonies could possibly be from pSB1K3 (i.e. if the selection didn't work properly), pEX.RFP 2 was also amplified with pSB primer VF2 and VR.
Also, pEX plasmid was amplified in parallel with pEX plasmid to compare the insert sizes.

  • J1: pEX.RFP 2
  • J8: pEX.RFP 3
  • J2: pEX.RFP 5
  • J3: pEX.RFP 6
  • J4: pEX clone
  • J5: pEX plasmid
  • J6: pEX.RFP 2 (w/ pSB primers)
  • J7: Blank

Procedures as described in colony PCR protocol:

  • Forward primer: pEXf or pSB VF2
  • Reverse primer: pEXr or pSB VR
  • Elongation: 2:00
Gel verification

To be added

Colony re-streak

In order to verify that our red colonies are indeed carrying pEX and not pSB1K3 (i.e. the Amp selection has failed), resulting in false-positives for transfer of RFP from pSB1K3 to pEX, clones 2, 3, 5 and 6 were streaked on quarters of Amp 100 and Km 50 plates. Plates incubated 48 h in 37 °C.
If RFP has been successfully transfered to pEX, the clones should only grow on Amp 100 plates.

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