Notebook : August 14

Lab work

A - Aerobic/Anaerobic regulation system

Obtaining the NarK or NarG or NirB _RBS-LacZ-term in PSB1C3

1 - Gel electrophoresis to check the digestion of Bba_K1155004,Bba_K1155005, Bba_K1155006 by EcoRI and SpeI

Anaïs, Nadia

Expected sizes :

• PSB1C33 : 2070kb
• NarK, NarG, NirB : 200kb

We obtained fragments of the right size but in very few quantity. We do it again but this time we will use more quantity of enzymes.

2 - Digestion of Bba_K1155004,Bba_K1155005, Bba_K1155006 by EcoRI and SpeI

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.

Obtaining RBS_LacZ+Term_PSB1C3

1 - Colony PCR on e.coli with RBS_LacZ+Term_PSB1C3 for 25 colonies

Anaïs

• Colony counting :
  • Low concentration petri dish : 47 colonies
  • High concentration petri dish : 145 colonies

• Picking of 25 colonies

• Preparation of 700µL of Master mix
  • H₂O : 590µL
  • dNTP : 28µL
  • VF2 primer : 3.5µL
  • VR primer : 3.5µL
  • DreamTaq buffer 10x : 70µL
  • DreamTaq enzyme : 5µL

Protocol : Colony PCR

PCR Program :

2 - Gel electrophoresis of the colony PCR products

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.