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)
<partinfo>BBa_C0060</partinfo>	Insert	16.5	4	1 EcoRI	1 SpeI	2.5	25
<partinfo>BBa_C0061</partinfo>	Insert	13.2	7.3	1 XbaI	1 PstI	2.5	25
<partinfo>BBa_K081022</partinfo>	Insert	15.7	4.8	1 EcoRI	1 PstI	2.5	25
<partinfo>BBa_B0030</partinfo>	Vector	13.2	7.3	1 SpeI	1 PstI	2.5	25
<partinfo>BBa_B0031</partinfo>	Vector	12.4	8.1	1 SpeI	1 PstI	2.5	25
<partinfo>BBa_B0032</partinfo>	Vector	9.5	11	1 SpeI	1 PstI	2.5	25
<partinfo>BBa_B0015</partinfo>	Vector	7.9	12.6	1 EcoRI	1 XbaI	2.5	25
<partinfo>pSB4C5</partinfo>	Vector	3	17.5	1 EcoRI	1 PstI	2.5	25
<partinfo>BBa_I13501</partinfo>	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 <partinfo>BBa_I13501</partinfo> run where we inexplicably didn't see any band), so we cut and purified the bands of interest. Cells harbouring <partinfo>BBa_I13501</partinfo> were again inoculated in 5 ml LB + Amp.

After gel extraction, cut DNA was quantified:

Plasmid	DNA (ng/μl)
<partinfo>BBa_C0060</partinfo> (E-S)	3.9
<partinfo>BBa_C0061</partinfo> (X-P)	4.1
<partinfo>BBa_K081022</partinfo> (E-P)	4.4
<partinfo>BBa_B0030</partinfo> (S-P)	10.3
<partinfo>BBa_B0031</partinfo> (S-P)	11.8
<partinfo>BBa_B0032</partinfo> (S-P)	10.1
<partinfo>BBa_B0015</partinfo> (E-X)	12
<partinfo>pSB4C5</partinfo> (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	<partinfo>BBa_B0015</partinfo> (E-X)	1.5	<partinfo>BBa_C0060</partinfo> (E-S)	6.5	1	1
E2	<partinfo>BBa_B0030</partinfo> (S-P)	1.5	<partinfo>BBa_C0061</partinfo> (X-P)	6.5	1	1
E3	<partinfo>BBa_B0031</partinfo> (S-P)	1.5	<partinfo>BBa_C0061</partinfo> (X-P)	6.5	1	1
E4	<partinfo>BBa_B0032</partinfo> (S-P)	1.5	<partinfo>BBa_C0061</partinfo> (X-P)	6.5	1	1
E8	<partinfo>pSB4C5</partinfo> (E-P)	1.5	<partinfo>BBa_K081022</partinfo> (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 <partinfo>BBa_I13501</partinfo> and purified DNA was quantified:

Plasmid	DNA (ng/μl)
<partinfo>BBa_I13501</partinfo>	97.2

<partinfo>BBa_I13501</partinfo> was then digested with restriction endonucleases:

Plasmid	Kind	DNA (μl)	H2O (μl)	Enzyme 1 (μl)	Enzyme 2 (μl)	Buffer H (μl)	Final Volume (μl)
<partinfo>BBa_I13501</partinfo>	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 <partinfo>BBa_I13501</partinfo> digestion:

Cut DNA was gel-extracted:

Plasmid	DNA (ng/μl)
<partinfo>BBa_I13501</partinfo> (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	<partinfo>BBa_B0030</partinfo> (S-P)	1	<partinfo>BBa_I13501</partinfo> (X-P)	7	1	1
E6	<partinfo>BBa_B0031</partinfo> (S-P)	1	<partinfo>BBa_I13501</partinfo> (X-P)	7	1	1
E7	<partinfo>BBa_B0032</partinfo> (S-P)	1	<partinfo>BBa_I13501</partinfo> (X-P)	7	1	1

July, 6th

<partinfo>BBa_C0261</partinfo> was resuspended from iGEM 2011 kit distribution, Plate 1, well 14C in 15 μl of ddH₂O; <partinfo>BBa_C0261</partinfo>, 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 (<partinfo>BBa_G00100</partinfo>) μl	VR (<partinfo>BBa_G00101</partinfo>) 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 and E8-3 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. <partinfo>BBa_R0040</partinfo> and <partinfo>BBa_C0261</partinfo> were also inoculated.

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