Notebook : August 8

Lab work

A - Aerobic/Anaerobic regulation system

((((((====Obtaining the PSB3K3 backbone plasmid====

1 - Electrophoresis of BBa_J004450 digested by EcoRI/Pst1 to check if the digestion was right

Damir, Nadia

Expected sizes :

• PSB3K3 : 2750kb
• GFP : 1069kb

We obtained fragments of the right size. Now we can purify the PSB3K3 plasmid.

2 - Gel purification of the PSB3K3 plasmid from BBa_J004450 digested by EcoRI/Pst1

2.1 - Migration of the remaining 45µL of BBa_J004450 digested by EcoRI/Pst1

Anaïs

Well 1 : 6µL DNA Ladder Well 2 : 45µL of BBa_J004450 digested by EcoRI/Pst1 Gel : 0.8%

Now we can do a gel purification of the highest band which contains the PSB3K3 plasmid.

2.2 - Electroelution of the highest band to extract the PSB3K3 plasmid from the gel

Nadia

Protocol : Electroelution

We let the plasmid precipitate during the night.))))))))))))))))))))))

Objective : obtaining Bba_K1155007

1 - Colony PCR of Bba_K115007 in DH5α

Anaïs

Colonie repiquée dans 10µL d'eau pour chaque tube ???????

Used quantities :

• DNA : 2µL
• Mix  : (it was divided in 25 tubes for each promotor with 23µL of mix in each on)
  • Oligo ... : 3.5µL
  • Oligo ... : 3.5µL
  • Buffer Dream Taq : 70µL
  • dNTP : 28µL
  • Dream Taq : 5µL
  • H2O : 590µL

PCR Program :

2 - Electrophoresis to check the colony PCR products : Bba_K1155007

Anaïs, Damir

6µL DNA Ladder 10µL sample per well Gel : 0.8%

Expected size : 3583bp

Colonies 10, 14, 15 exhibit plasmids with the right length.

3 - PCR product (made the 08/01/2013) purification

Damir

available quantity:

• FNR Part1 : 10 µl
• FNR Part2 : 19 µl
• RBS FNR Part1 :16.1µl
• RBS BphR2 Part1 : 28µl
• BphR2 Part1 : 16.4 µl
• BphR2 Part2 : 18.9 µl

Protocol : kit purification

Manipulation error : The elution step was made using the recuperation tube from the filtering step, instead of a new, clean eppendorf tube.