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.