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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