Week 12

08/17 - 08/21

Gibson Assembly

• Gel Migration of the PCR products (Cluster 2 and BBa_808014)
• PCR Amplification of intersequence 936011/936023 using Takara Ex Taq Polymerase.
• Gel Migration on 0,8% Agarose Gel
• PCR Amplification of BBa_316003 using Takara Ex Taq Polymerase.
• Gel Migration using 0,8% Agarose Gel
• Spe I and Pst I Restriction Digest of the recieved plasmids containing BBa_K936011, BBa_K13932932, K936023.
• Gel Purification of the digested plasmids K936011 and K1392932 using the QIAgen Gel extraction kit protocol.

NB -Esterase assay

• Liquid culture of the second transformation (of the 15/08/15): BBa_808030 and pDG011 plasmid in DH5α
• MiniPrep of BBa_808030 in the plasmid pDG011 in BAP 1
• XbaI and PstI digestion of BBa_808030 and pDG011 (with the phosphatase step)

Gel Purification

Tubes components:

• Ligation of BBa_808030 and pDG011
• Transformation in Chemically Competent DH5-⍺ cells
• Transformation in DH5-ɑ of the biobrick K808030 in pDG011
• Transformation in DH5α cells of BBa_808030 insert and pDG011 vector
• SpeI and Pst I Enzymatic Digest of BBa_K808030 with dephosphorylation.
• Gel migration
• Gel extraction of the PCR product of K808030 and pDG011.
• Ligation of pDG011 and K808030.

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 50mL of BBa_J61047 culture. (dilution 1/1000)

• 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 » : 5uL 10X buffer cutsmart + 0,5uL XbaI + 0,5uL SpeI + 38uL H2O +5uL ADN, digestion at 37°C during 1 hour.
• tube 2 « Midiprep XbaI/SpeI » : 5uL 10X buffer cut smart + 0,5uL XbaI + 0,5uL SpeI + 38uL H2O + 5uL ADN, digestion at 37°C during 1 hour
• tube 3 « Miniprep NotI » : 5uL 10X buffer cutsmart + 0,5 NotI + 38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour
• tube 4 « MiDiprep NotI » : 5uL 10X buffer cutsmart + 0,5 NotI + 38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour
• tube 5 « Miniprep CTRL » as negative control : 5Ul ADN + 40uL H2O + 5uL 10X buffer cutsmart.
• tube 6 « Midiprep CTRL » as negative control : 5Ul ADN + 40uL H2O + 5uL 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 (See figure 01)
  
• Culture of the plasmid pRS415 in SC_Leu at 30°C at 150rpm for about 72h.
• PCR Amplification using the Phusion Polymerase of pRS415.

NB-Esterase Assay:

	Concentration (ng/µL)	OD(230nm)	OD(260nm)	OD(280nm)	OD(260nm)/OD(230nm)	OD(260nm)/OD(280nm)
Cluster_4_start	30	0,025	0,575	0,375	1,50	-
/936011	55	0,4	1,075	0,575	1,88	2,65
936011/936023	7,5	0,6	0,125	0,05	2,41	0,21
936023/	45	0,575	0,925	0,625	1,48	1,61
Cluster_4_end	47,5	0	0,925	0,575	1,64	-

Gel plan:

Stain	1	2	3	4	5	6	7	8	9	10	11
Tube number			A1	A2	A3	A4		B1	B2	B3	B4
Componants	Ladder

                                                                      

 

	Concentration (ng/µL)	OD(230nm)	OD(260nm)	OD(280nm)	OD(260nm)/OD(280nm)	OD(260nm)/OD(230nm)
Cluster 4 start	105	0,3	2,1	1,1	1,79	2,40
/936011	55	0	1,1	0,55	1,99	-
936011/936023	520	4,325	10,425	5,825	1,79	4,38
936023/	47,5	0	0,925	0,5	1,89	-
Cluster 4 end	130	0,6	2,625	1,475	1,79	4,38

DNA concentration assay

pNP-Assay

1. Culture of DH5-alpha transformed with 808030 in pDG011
2. Enzymatic essay (pNP assay) of the first transformation (of the 11/08/15): BBa_808030 and pDG011 plasmid in DH5α

Gibson Assembly :
Enzymatic digest of K936011, K936023 and K1392932

1. Enzymatic digest using Pst I and Spe I
2. Gel Migration of digested plasmids K936011, K936023, K1392932. on a 0,8% Agarose Gel
3. Gel Purification

Cluster 4A Gibson assembly :

1. Gibson Assembly
2. PCR amplification of cluster 4A Assembly.
3. Gel migration on 0,8% Agarose Gel.

Gel Plan:

Well Number	1	2	3	4	5
Contains	DNA ladder	No primers	Primer for only	Primer rev only	Both primers





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