Team:Warsaw/Calendar-Main/1 October 2008

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Preparation of vectors for Biobricks

Michał K., Piotr

  1. Isolation of pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids.
  2. Digest of isolated plasmids with EcoRI and BcuI (BamHI buffer). Dephosphorylation (CIAP) of plasmids.
  3. Gel elctrophoresis and gel-out of proper bands: 4500 bp - pSB2K3 and 3000 bp - BBa_I739204 (pACYC177 converted into BioBrick vector).
Fig. 1.Digest of pSB2K3 and BBa_I739204 with EcoRI and BcuI
1. Marker
2. Digested pSB2K3
3. Digested BBa_I739204

Preparation of Z(BBa_K103004)

Michał K.

Inoculation of some colonies from pSB1A3+Z(BBa_K103004) plate to LB with ampicillin.

Preparation of OmpA_linker_omega_linker under Plac (BBa_K103018)

Michał K.

  1. Addition of 5 μl T4 ligase buffer and 2 μl of T4 ligase to digest of BBa_K103018 fragment. Ligation for 1 hr.
  2. PCR on ligation of OmpA_linker_omega_linker under Plac (BBa_K103018) fragment using PlacL_XNE and LinP_BS primers (annealing temperature 58 °C; elongation length 90s) to obtain BBa_K103018 fragment without internal EcoRI site (Przanowski' method of removing internal restriction site).
  3. Gel electrophoresis of PCR product and gel-out of proper bands (BBa_K103018 - 1200 bp).
  4. Digest of PCR product with XbaI and PstI (Tango buffer).
  5. Clean-up of above digest reaction.

Preparation of AID(BBa_K103001)

Michał K.

  1. PCR on pMPMT5+AID plasmid using AIDl and AIDP_HindSpeNotPst primers (annealing temperature 58 °C; elongation length 60s) to obtain AID(BBa_K103001) fragment.
  2. Gel electrophoresis of PCR product and gel-out of proper bands (AID(BBa_K103001) - 600 bp).
  3. Digest of PCR product with XbaI and PstI (Tango buffer).
  4. Clean-up of above digest reaction.
Fig. 2. PCR to obtain AID(BBa_K103001)
1. Marker
2. AID(BBa_K103001)
3. ???