User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/07/07

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July 7th, 2010

1. Plasmid extraction using the High Pure Plasmid Isolation Kit Roche.

  • Incubate on 4 ml of liquid LB medium the transformations made with ligations 1-6 to extract plasmid, transformants contain the following plasmids:
1. p30-MinBP + GFP E0240
2. p30-MinBP + GFP BBa_K145015 (74 min)
4. Backbone plasmid pSB3K3 + Blue Promoter BBa_K238013 + GFP BBa_K145015 (74 min)
5. Plasmid 18 pSB1T3 + GFP BBa_K145015
6. Religation p30-MinBP to make it a standard BioBrick
  • I will also extract plasmid 18, 19 & 30 to store as stock for future uses.
  • Ligation 3 Backbone plasmid pSB3K3 + Blue Promoter BBa_K238013 + GFP E0240 was not successful, so I didn’t extracted plasmid. I’ll extract the Blue Promoter BBa_K238013 + GFP E0240 from the ligation 1 p30-MinBP + GFP E0240 by PCR and then ligate to the backbone plasmid.


2. Restriction to verify ligations from extracted plasmids

Restriction with PstI (DNA 5 ul, Total volume 20 ul):
DNA -> 5 ul
Buffer 3 -> 2 ul
BSA -> 1 ul
PstI -> 1.5 ul
HPLC -> 10.5 ul
Incubate at 37ºC overnight
Double restriction methods EcoRI-HF and PstI (DNA 5 ul, Total volume 20 ul):
DNA -> 5ul
Buffer 2 -> 2ul (10% of total volume)
BSA (required by PstI) -> 1ul
EcoRI-HF -> 1ul
PstI -> 1ul
HPLC -> 10ul (to complete total volume of 20ul)
Incubate at 37ºC overnight



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