Team:Warsaw/Calendar-Main/1 October 2008

From 2008.igem.org

Gallery Bricks Notebook Team Project Home


Previous day
return to main notebook page
Previous entry
next notebook entry

 


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.
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's method of removing internal restriction site).
  3. Gel electrophoresis of PCR product and gel-out of proper bands (BBa_K103018 - 1200 bp). Fig. 2.


Fig. 2. PCR to obtain AID and OmpA_linker_omega_linker under Plac
1. Marker
2. AID
3. OmpA_linker_omega_linker under Plac

Preparation of AID(BBa_K103001)

Michał K.

  1. PCR on pMPMT5+AID plasmid using AIDl and AIDP_SpeNotPst 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). Fig. 2.
  3. Digest of PCR product with XbaI and PstI (Tango buffer).
  4. Clean-up of above digest reaction.
Fig. 2. PCR to obtain AID and OmpA_linker_omega_linker under Plac
1. Marker
2. AID
3. OmpA_linker_omega_linker under Plac