Week 3

26/07/2015

• The 3 transformation tubes containing Promoter2 + Ampicillin, Promoter3+Ampicillin, Promoter1+ Ampicillin, are still clean so we proceeded to discard them.
• After centrifuging the tubes, all the Terminator in Ampicillin tubes have white pellets except Ter2 tube (diluted), which had a red pellet. Hence we discarded Ter2 tube.
• We performed the Miniprep on the other 5 tubes with white pellets.
• Then performed digestion and ligation on the plasmids using standard protocols.
• Then we transformed the DNA into competent cells

Ligation

• Exception for the ligation:
  1. We added 5ul of each digest and ccdb backbone
  2. 2ul of DNA ligase
  3. 4ul of buffer solution but no water
• This was spun down for 10 seconds in the centrifuge.

Transformation:

• The transformation was done according to standard protocol
• However, for step 7, tube 1& 3 are placed in the normal incubator at 37˚C and at 130rpm
• However, for step 7, tube 1& 3 are placed in the normal incubator at 37˚C and at 130rpm
• We created 2 duplicates of transformation plate for each tube.

27/07/2015

• We did not use the correct restriction enzymes on the ccdb cells; hence we had to re-do the ligation and digestion of the plasmids. See above for the protocol ( for the 26/07/15)
• We then followed with the transformation of the plasmids by transforming 5ul each of the transformed plasmids in each tube of the competent cell

Transformation:

The transformation was followed according to standard protocol, with the exception of certain steps:

1. Heat shock the cells at 42˚C.
2. The cells were placed on ice for 3 instead of 5 minutes
3. Pipette 900ul of S.O.C. mixture
4. The tubes were shaken in the heating block at 37˚C, at 250rpm.

28/07/2015

For the stock concentration of antibiotic:

• we need 50ug/mL of Chloroamphenciol ( from earlier documentation)
• target conc: 50uh/mL
  target conc: 50uh/mL
• So we have 300ml of LB and our final conc. needs to be 50ug/mL. Hence we measure out 6mg of antibiotic into the broth.
• So We added 330ul of antibiotic ( of stock concentration 100mg/mL) into 300ml of LB Broth, then pipetted 10ml into each test tube.
• Picked colonies from each plate, inoculated them and left them in the incubator at 250rpm.

29/07/2015

• Miniprepped the plasmids but we accidentally threw out 2 test tubes of DNA but we still had 4 duplicates left.
• We then performed a Nanodrop of the ChiA constructs

26/08/2015

• Performed ligation of the promoter and tnaA
• Amplifying the tnaA and tnaB sequence.
• Added 200ul of water (DNA- RNA free) into each tube
• Performed the Nano Drop . The concentration of both tubes is approximately 7-8ug/ul.

27/08/2015

• Nanodrop to verify concentration of resuspended DNA
• Nuclease- free H2O used to resuspend the DNA was found to be contaminated with 4.8ug/ul of DNA
• The actual DNA content of plasmids are around 6-8 ug/ul on average
• We decided to proceed with the transformation since protocol specified 10ug- 100ug of DNA.

30/08/2015

• We made liquid broth, Kanamycin

Preparing agar plates:

1. Take 35g of agar powder in 1L of solution.
2. Add the powder then autoclave the solution( 1hour)
3. We already have antibiotic stock solution of 100mg/ml

Note: Due to coagulation, we had to put in 400ul of antibiotic( chloroamphenicol) instead of 500ul as originally desired.