Week 12

08/17 - 08/21

NB-Esterase Assay:

MiniPrep 808030 in the plasmid pDG011.

pNP-Assay:

• Culture of DH5-α transformed with 808030 in pDG011
• Enzymatic assay (pNP assay) of the first transformation (of the 11/08/15): BBa_808030 and pDG011 plasmid in DH5-α

Gibson Assembly:

OD measurement of Cluster 4:

• Gel migration of the PCR product of prom/pNB Est and Cluster_1_start.

Gel plan:

DNA concentration assay :

Gibson Assembly:

• Enzymatic digest of K936011, K936023 and K1392932
• Enzymatic digest using Pst I and Spe I
• Gel Migration of digested plasmids K936011, K936023, K1392932. on a 0.8% agarose gel
• Gel Purification

Cluster 4A Gibson assembly:

• Gibson Assembly
• PCR amplification of cluster 4A Assembly.
• Gel migration on 0.8% agarose gel.

Gel Plan:

PCR amplification of Biobricks K808007, K808030, K936011, K936023, K1392932, K316003

• PCR amplification using Phusion polymerase
• Gel Migration on a 1% agarose gel

The PCR worked for most of our Biobricks with the exception of the biobrick K808030.

DNA concentration measurement

PCR amplification of pSB1C3

• PCR amplification using Phusion Polymerase.
• Gel Migration on a 1% agarose gel

PCR amplification of pSB1C3

• PCR amplification using Takara Ex Taq Polymerase testing different annealing temperatures.

Cycles:

• 95°C for 4min
• 25 cycles :
• 95°C for 40s
• annealing for 30s:
• 60°C: Tube 1-4
• 61.1°C: Tube 5-8
• 63°C: Tube 9-12
• 65.6°C: Tube 13-16
• 69.2°C: Tube 17-20
• 72.1°C: Tube 21-24
• 73.9°C: Tube 25-28
• 75°C: Tube 29-32
• 72°C for 80s
• 72°C for 7min.




PCR amplification using Takara Ex Taq

• Gel Migration on a 1% agarose gel.

PCR amplification of Biobricks K808030

• PCR Amplification using Takara Ex Taq Polymerase.
• Gel migration on 1%



Gel Plan:

Well Number	1	2	3	4	5
	DNA ladder	No primers	Primer For	Primer Rev	Extract





PCR amplification of pSB1C3

• PCR amplification using Takara Ex Taq Polymerase testing different annealing temperatures.
• Gel Migration on a 1% agarose gel.

PCR amplification of pSB1C3

• PCR amplification using Takara Ex Taq Polymerase testing 8 annealing temperatures.

	No primers	Primer for	Primer rev
DNAse, RNAse free water	41.75	40.75	40.75	39.75
10X Ex Taq Buffer	5	5	5	5
dNTP	2	2	2	2
rescue_for	0	1	0	1
rescue_rev	0	0	1	1
DNA Template	1	1	1	1
Ex Taq Polymerase	0.25	0.25	0.25	0.25
Total	50	50	50	50




Gel Migration on a 1% Agarose Gel

Enzymatic digest of BBa_K808030

• Enzymatic digest by XbaI and Spe I of BBa_K808030
• Gel migration.



Yeast Assembly

The bacteria did not grow because the wrong antibiotic was chosen: BBa_J61047 was in pSB1A2 and not pSB1C3.

• Bacterial culture of BBa_J61047 on liquid LB + Amp 1X at 180 rpm, 37°C. The bacteria grew well.
• Midi-prep has been done with the 50 mL of BBa_J61047 culture. (dilution 1/1000)

	DNA Concentration (ng/µl)	OD(260nm)	OD(280nm)	OD(260/280nm)
BBa_J61047	0.5	0.011	0.004	2.57



• Preparation of YPD specific media on specific media (1% yeast extract, 2% dextrose, 2% peptone, 2% agar).
• Gel Migration of BBa_J61047.
• NotI enzymatic digest of BBa_J61047 in pSB1A2 enzyme.
• Gel Migration on a 0.8% agarose gel and the digestion hasn’t work.
• XbaI and SpeI digestion of BBa_J61047.
• tube 1 « Miniprep XbaI/SpeI » : 5 µL 10X buffer cutsmart + 0.5 µL XbaI + 0.5 µL SpeI + 38 µL H₂O +5 µL DNA, digestion at 37°C during 1 hour.
• tube 2 « Midiprep XbaI/SpeI » : 5 µL 10X buffer cut smart + 0.5 µL XbaI + 0.5 µL SpeI + 38 µL H₂O + 5µL DNA, digestion at 37°C during 1 hour
• tube 3 « Miniprep NotI » : 5 µL 10X buffer cutsmart + 0.5 µL NotI + 38.5 µL H₂O + 5 µL DNA, digestion at 37°C during 1 hour
• tube 4 « MiDiprep NotI » : 5 µL 10X buffer cutsmart + 0.5µL NotI + 38.5µL H₂O + 5 µL DNA, digestion at 37°C during 1 hour
• tube 5 « Miniprep CTRL » as negative control : 5 µL DNA + 40 µL H₂O + 5 µL 10X buffer cutsmart.
• tube 6 « Midiprep CTRL » as negative control : 5 µL DNA + 40 µL H₂O + 5 µL 10X buffer cutsmart.
• MWT : « Quick-Load Purple 2-Log DNA Ladder (0.1-10.0 kb) NEB




PCR Amplification of the BBa_J61047 using Phusion Polymerase Master Mix.

• Gel Migration on a 0.8% agarose gel
• Culture of the plasmid pRS415 in SC_Leu at 30°C at 150 rpm for about 72h.
• PCR Amplification using the Phusion Polymerase of pRS415.
• Gel Extraction of the PCR products. 




TPA solubility :

• Determination of the solubilization products of TPA.
• Detection of 2-hydroxy-terephthalate using a TECAN spectrometer.
• Precipitaion of terephthalic acid in H₂SO₄.




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