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

Gel Migration on a 1% Agarose Gel

As you can see, the PCR worked for most of our Biobricks with the exception of the biobrick K808030.

1. DNA concentration measurement

1/25 dilution	DNA concentration (ng/µL)	OD(230nm)	OD(260nm)	OD(280nm)	OD(340nm)	OD(260nm)/OD(280nm)	OD(260nm)/OD(230nm)
808007	4,3	0,08	0,085	0,052	0,006	1,64	1,06
808030	4,6	0,151	0,092	0,058	0,003	1,59	1,61
936011	5,9	0,141	0,118	0,073	0,009	1,61	0,83
936023	5,2	0,097	0,105	0,062	0,003	1,7	1,08
1392932	12,6	0,493	0,251	0,259	0,035	0,97	0,51
316003	5,5	0,1	0,11	0,062	0,0	1,76	1,10

PCR amplification of pSB1C3

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

Well Number	1	2	3	4	5	6	7
Contains	DNA ladder	Non digested Plasmid	Digested plasmid	No primers	Primer for only	Primer rev only	Both primers

 

PCR amplification of pSB1C3
- PCR amplification using Takara Ex Taq Polymerase testing different 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

Cycles:

1. 95°C for 4min
2. 25 cycles :
1. 95°C for 40s
2. annealing for 30s:
1. 60°C: Tube 1-4
2. 61,1°C: Tube 5-8
3. 63°C: Tube 9-12
4. 65,6°C: Tube 13-16
5. 69,2°C: Tube 17-20
6. 72,1°C: Tube 21-24
7. 73,9°C: Tube 25-28
8. 75°C: Tube 28-32
3. 72°C for 80s
3. 72°C for 7min.




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