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Team:UNAM-Genomics Mexico/Notebook/pBBR1MCS-5
From 2011.igem.org
Contents |
Lab Logbook -pBBR1MCS.
Week: june 13 to 18
At beginning of the week was decided the best way to insert a prefix-RFP-suffix fragment to thepBBR1MCS-5 plasmid, which is a plasmid with broad host-range and is capable to conjugate to R. etli through E. coli S17, being the most efficient way to introduce genes into R. eltli. The pBBR1MCS-5 has a multiple cloning site (MCS) with the following restriction sites:
KpnI
ApaI
XhoI
Sal I
Bsp106 I
ClaI
HindIII
EcoR1
PstI
SmaI
Bam H1
SpeI
XbaI
BstXI
SacI
So we want to eliminate the most of the restriction sites and obviously we need to eliminate the prefix and suffix sites (italic). To do it we are going to cut in the ApaI and SacI site, on the other hand we're going to amplify a region which contains the suffix, an RFP and the prefix of the plasmid pSB1T3, this PCR amplification will be done with primers containing ApaI and SacI respectively, thereafter we'll can insert this amplification in our pBBRMCS-5.
Once decided the way to add the prefix and suffix to pBBR1MCS-5, we spent the rest of the week isolating the plasmid and trying to digest the plasmid by apaI, knowing that only works with strains dcm-. Knowing that E. coli DH5α is dsc+ we tried unsuccessfully to cut with ApaI, the following gel shows it:
[http://openwetware.org/images/0/08/Gel_18_jul_2011.png]
