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Revision as of 22:37, 17 September 2015

Week 5

30/08/15

  • We made liquid broth and added Kanamycin and Amp, 500ml for each ( see standard procedures)
  • noculated individual colonies in LB broth with respective antibiotic of tnaa and tnab. However, we accidentally switched antibiotics so there was only 1 tube of each of the parts. Left to incubate in shaking incubator at 37˚C.

31/08/2015

  • Miniprepped the tnaA and tnaB cultures from IDT (see standard QIAprep protocol)
  • The large centrifuge was broken, so the cultures were spun down in Eppendorf tubes per large tube.
  • Completed a Nanodrop . Results shown below:
  • Completed the digestion and ligation of the tnaA
  • Transformed the cells

28/08/2015

  • We received the tnaa and the tnab tubes from IDT on the 26/08/15
  • Resuspended the tnaa and the tnab sequence by adding 200ul of water ( DNAse and RNAase free) into each tube.

29/08/2015

  • Nanodropped the DNA to verify the concentration of resuspended DNA. The Nuclease-free water that was used to reuspend the DNA was found to be contaminated with 4.8ng/ul of DNA. The actual DNA content of the plasmids is around 6-8ng/ul on average.
  • We still proceeded with the transformation of the DNA since the protocol specified 10ug-1000ug of DNA.
Results Of Nano Drop
Sample Concentration Unit A260 A280 260/280 260/320
tnaA 9.9 ng/ul 0.198 0.085 2.33 1.26
tnaA_2 9.7 ng/ul 0.194 0.082 2.36 1.22
tnaB 25.4 ng/ul 0.507 0.266 1.91 1.09
tnaB_2 22.9 ng/ul 0.459 0.227 2.02 1.21

Digestion:

  • Aim: To put the promoter and RBS upstream of tnaA

PHOTO

Digestion:

  • double digest, total volume- 50uL ( followed standard protocol exactly)

Ligation:

  • Triple ligation, total vol. -20uL:
    • 5ul of digest x3 ( upstream, downstream, destination plasmid)
    • 2uL of 10x buffer
    • 1ul of T4 ligase
    • 2ul of water
  • Remainder of the protocol was standard protocol of ligation

Transformation:

  • Followed the Eb-5α protocol exactly (heat shocked the cells at 42˚C for 45 seconds)
  • Did duplicate tubes, streaking on separate plates.

02/09/15

  • Checked the plates, but there were very few white colonies present, so we discarded them
  • Hence, we redid the digestion and ligation of tnaA (standard protocol, see 31/08/15) to be sure.

Digestion:

  • Promoter + RBS – cut with EcoRI + Spel
  • TnaA- cut with Xbal + PstI
  • RFP backbone – cut with EcoRI + PstI

Ligation:

  • We made duplicates
  • Verified the ligation by running it through electrophoresis gel before transforming.
    • Gel: agarose, 100V
    • Lanes L-R: T1, T2, RFP, ladder. The T1 and T2 sample bands were slower than RFP. Proceeded with transformation

Transformation:

  • transformed in duplicate ( 2 tubes) up to outgrowth step
  • The agar plates in the incubator. 2 plates were placed in the incubator and 2 were placed in the shaking incubator.

03/09/15

  • Checked the plates, there was no growth. Redoing transformation.
    • 4 tubes, 2.5uL of ligation mixture each
    • Heat shock at 42˚C of 45seconds
    • 4 tubes in shaker.

03/09/15

  • Out of the 4 tubes, 3 were found to be red, only 1 white.
  • Hence, we miniprep the 1 tube. We also streaked a plate with the culture from the tube, in case the miniprep does not work.
  • We also replated some colonies by inoculating them in liquid culture for 30mins and streaking them onto plates, in hope of getting some white colonies. There were 3 plates, labelled with blue marker, placed in the incubator at 1146h
Results Of Nano Drop Of miniprep
Tube Nucleic acid conc. Unit A260 A280 260/280 260/320
Miniprep 1 3.1 ng/ul 0.062 0.019 3.19 3.19
Miniprep 2 3.9 ng/ul 0.078 0.027 2.93 2.13

The results show that while the ratio of DNA to other substances is high ( i.e. the DNA is pure), there is very little amount of DNA in the minipreps.
We also inoculated the lldd from IDT that we received.