User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/06/25

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June 25th, 2010

1. Run gel to verify PCR from June 24th.

PCR p30-MinBP 24Jun2010.JPG

Lanes: 1) p30-MinBP Jorge’s reactives (JR) Tube 1; 2) p30-MinBP Jo rge’s reactives (JR) Tube 1; 3) Ctrl ~3 kb’s JR Tube 2; 4) Ctrl - JR Tube 3; 5) p30-MinBP Paz’s reactives (PR) Tube 4; 6) Ctrl ~3 kb’s PR Tube 5; 7) Ctrl - PR Tube 6; 8) p30 1:4 dilution; 9) Ladder.

  • PCR to amplify p30-MinBP was succesful with Paz’s reactives but not with mines, Ctrl ~3 kb’s was amplified very weakly with Paz’s reactives and with mines, so I think my MinBP Rev and Suffix Fwd primers are not working well.

2. Extract backbone plasmid following the High Pure Plasmid Isolation Kit Roche.

  • Plasmid extraction was performed by duplicate, tubes were marked: “Backbone plasmid pSB3K3 1 25.JUN.10” and “Backbone plasmid pSB3K3 2 25.JUN.10”.

3. Restriction to backbone plasmid (pSB3K3) just extracted with EcoRI and PstI.

Double restriction methods EcoRI and PstI:

DNA -> 5ul
Buffer 4 -> 2ul (10% of total volume)
BSA (required by SpeI) -> 1ul
EcoRI -> 1ul
SpeI -> 1ul
HPLC -> 10ul (to complete total volume of 20ul)
Incubate at 37ºC overnight

4. Purify PCR product from June 22nd (rTth p30-MinBP made by Paz) and June 24th (p30-MinBP Paz’s reactives, Tube 4).

  • PCR product purification was made using the High Pure PCR Product Purification Kit Roche.
  • Purified PCR products were joined in one tube marked: “p30-MinctBP rTth purified 25.JUN.10” and “p30-MinBP Platinum Taq purified 25.JUN.10”

5. Run gel to verify extracted Backbone plasmid pSB3K3, the restrictions made to this plasmid and the purified and joined PCR products of p30-MinBP.

Backbone plasmid extracted, restrictions and purified PCR product p30-MinBP.JPG

Lanes: 1) Ladder; 2) Backbone pSB3K3 1; 3) Backbone pSB3K3 2; 4) EcoRI-PstI Backbone pSB3K3 1; 5) EcoRI-PstI Backbone pSB3K3 2; 6) p30-MinBP rTth purified; 7) p30-MinBP Platinum Taq purified.

6. Restriction to p30-MinBP rTth purified with SpeI and PstI-SpeI.

Restriction with SpeI (DNA 10 ul, Total volume 30 ul):

DNA -> 10 ul
Buffer 4 -> 3 ul
BSA -> 1 ul
SpeI -> 1.5 ul
HPLC -> 14.5 ul
Incubate at 37ºC overnight

Double restriction methods SpeI and PstI (DNA 10 ul, Total volume 30 ul):

DNA -> 10 ul
Buffer 4 -> 3 ul (10% of total volume)
BSA (required by SpeI) -> 1 ul
PstI -> 1.5 ul
SpeI -> 1.5 ul
HPLC -> 13 ul (to complete total volume of 30ul)
Incubate at 37ºC overnight