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Revision as of 22:35, 17 September 2015

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
Results Of 2nd Nano Drop
Tube Nucleic acid Unit A260 A280 260/280 260/320 Factor
ChiA 3.1 95.2 ng/ul 1.904 0.987 1.93 2.27 50
ChiA 2.1 37.0 ng/ul 0.741 0.379 1.96 1.82 50
ChiA 2.2 360.0 ng/ul 7.200 3.811 1.89 2.26 50
ChiA 3.2 156.3 ng/ul 3.127 1.646 2.02 1.91 50

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.