Team:UNIPV-Pavia/Calendar/July/settimana2

July, 4th

Digestions of previously purified plasmids were performed for ligations:

Plasmid	Kind	DNA (μl)	H2O (μl)	Enzyme 1 (μl)	Enzyme 2 (μl)	Buffer H (μl)	Final Volume (μl)
BBa_C0060	Insert	16.5	4	1 EcoRI	1 SpeI	2.5	25
BBa_C0061	Insert	13.2	7.3	1 XbaI	1 PstI	2.5	25
BBa_K081022	Insert	15.7	4.8	1 EcoRI	1 PstI	2.5	25
BBa_B0030	Vector	13.2	7.3	1 SpeI	1 PstI	2.5	25
BBa_B0031	Vector	12.4	8.1	1 SpeI	1 PstI	2.5	25
BBa_B0032	Vector	9.5	11	1 SpeI	1 PstI	2.5	25
BBa_B0015	Vector	7.9	12.6	1 EcoRI	1 XbaI	2.5	25
pSB4C5	Vector	3	17.5	1 EcoRI	1 PstI	2.5	25
BBa_I13501	Insert	7.2	13.3	1 XbaI	1 PstI	2.5	25

Reactions were incubated at 37°C for three hours while a small-size and a medium-size agarose gel were prepared according to protocols.

In the afternoon gel electrophoresis was performed.

As shown, in figure all clones were positive (apart from BBa_I13501 run where we inexplicably didn't see any band), so we cut and purified the bands of interest. Cells harbouring BBa_I13501 were again inoculated in 5 ml LB + Amp.

After gel extraction, digested DNA was quantified:

Part	DNA (ng/μl)
BBa_C0060 (E-S)	3.9
BBa_C0061 (X-P)	4.1
BBa_K081022 (E-P)	4.4
BBa_B0030 (S-P)	10.3
BBa_B0031 (S-P)	11.8
BBa_B0032 (S-P)	10.1
BBa_B0015 (E-X)	12
pSB4C5 (E-P)	10.7

Then ligations were performed in a final volume of 10 μl:

Ligation Name	Vector	Vector volume (μl)	Insert	Insert volume (μl)	Buffer (μl)	T4 Ligase (μl)
E1	BBa_B0015 (E-X)	1.5	BBa_C0060 (E-S)	6.5	1	1
E2	BBa_B0030 (S-P)	1.5	BBa_C0061 (X-P)	6.5	1	1
E3	BBa_B0031 (S-P)	1.5	BBa_C0061 (X-P)	6.5	1	1
E4	BBa_B0032 (S-P)	1.5	BBa_C0061 (X-P)	6.5	1	1
E8	pSB4C5 (E-P)	1.5	BBa_K081022 (E-P)	6.5	1	1

Ligations were incubated ON at 16°C.

July, 5th

T4 Ligase was inactivated, heating ligations at 65°C for 10 minutes; ligations were stored at -20°C. Plasmid purification was done again for BBa_I13501 and purified DNA was quantified:

Plasmid	DNA (ng/μl)
BBa_I13501	97.2

BBa_I13501 was then digested with restriction endonucleases:

Plasmid	Kind	DNA (μl)	H2O (μl)	Enzyme 1 (μl)	Enzyme 2 (μl)	Buffer H (μl)	Final Volume (μl)
BBa_I13501	Insert	10.5	10	1 XbaI	1 PstI	2.5	25

According to protocols the reaction was incubated at 37°C for 3 hours.
60 ml of 80% glycerol were prepared mixing 48 ml of 100% glycerol with 12 ml of ddH₂O.
Gel electrophoresis was done for BBa_I13501 digestion:

Digested DNA was gel-extracted:

Part	DNA (ng/μl)
BBa_I13501 (X-P)	2.6

Further ligations were prepared and incubated ON at 16°C:

Ligation Name	Vector	Vector volume (μl)	Insert	Insert volume (μl)	Buffer (μl)	T4 Ligase (μl)
E5	BBa_B0030 (S-P)	1	BBa_I13501 (X-P)	7	1	1
E6	BBa_B0031 (S-P)	1	BBa_I13501 (X-P)	7	1	1
E7	BBa_B0032 (S-P)	1	BBa_I13501 (X-P)	7	1	1

July, 6th

BBa_C0261 was resuspended from iGEM 2011 kit distribution, Plate 1, well 14C in 15 μl of ddH₂O; BBa_C0261, E1, E2, E3, E4, E5, E6, E7, and E8 were transformed in 100 μl of TOP10 competent cells according to protocols. Plates were incubated ON at 37°C.
500 ml of LB without antibiotic were prepared.

July, 7th

All plates showed a lot of colonies, except for E-8 (5 colonies); two colonies for E1, E2, E3, E4, E5, E6, E7 plates and three colonies for E8 plate were picked and inoculated in 10 μl LB for inoculum and screening PCR.
A 20x mix was prepared for PCR reaction:

H2O (μl)	Buffer 10x (μl)	MgCl2 (μl)	VF2 (BBa_G00100) μl	VR (BBa_G00101) μl	dNTPs (μl)	Taq polymerase (μl)
360	50	20	10	10	10	20

24 μl were aliquoted in each tube, together with 1 μl of each liquid culture. PCR cycles parameters were set as follows:

• 94°C 30 seconds (denaturing)
• 60°C 1 minute (annealing)
• 72°C 1 minute and 20 seconds (elongation)

35 cycles were performed.
Liquid cultures were then inoculated in 800 μl LB with the proper antibiotic.
A small size and a medium size agarose gel were prepared.

After the PCR, gel run was carried out on the samples:

Except for E2-1, E4-1 and E7-1, other band lengths were correct; 750 μl of E1-2, E2-2, E3-1, E4-2, E5-2, E6-1, E7-2, E8-3 and BBa_C0261 liquid cultures were used to prepare glycerol stocks while the remaining 50 μl were refilled to 5 ml for plasmid purification and incubated at 37°C 220 rpm. BBa_R0040, BBa_C0261 and BBa_I13521 were also inoculated.
In the late afternoon the team met to talk about the wet-lab activity, the abstract and the bio-safety section.

July, 8th

Cultures were grown till saturation; plasmid purification was carried out:

Plasmid	DNA (ng/μl)
E1-2	64.1
E2-2	56.2
E3-1	53.5
E4-2	67.2
E5-2	73.5
E6-1	92.2
E7-2	67.6
E8-3	25.3
BBa_R0040	48.1
BBa_C0261	61.2
BBa_I13521	79.3

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