Team:UNIPV-Pavia/Calendar/July/settimana4

July, 18th

BBa_R0040 in BBa_J61002 plasmid purification and quantification was carried out:

Plasmid	DNA (ng/μl)
BBa_R0040 in BBa_J61002	37.3

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)
E9-2	Insert	11.5	9	1 Xbal	1 Pstl	2.5	25
E10-1	Insert	10	10.5	1 Xbal	1 Pstl	2.5	25
E11-1	Insert	4	16.5	1 Xbal	1 Pstl	2.5	25
E12-1	Insert	10	10.5	1 Xbal	1 PstI	2.5	25
E13-1	Insert	6	14.5	1 EcoRl	1 Pstl	2.5	25
E16-1	Insert	4	16.5	1 EcoRl	1 Pstl	2.5	25
E21-1	Insert	7.5	13	1 EcoRl	1 Pstl	2.5	25
E22-2	Insert	10	10.5	1 EcoRl	1 Pstl	2.5	25
BBa_I13521	Insert	12.5	8	1 EcoRl	1 Pstl	2.5	25
BBa_R0040 in BBa_J61002	Vector	20.5	0	1 EcoRl	1 Pstl	2.5	25

Reactions were incubated at 37°C for three hours; meanwhile two medium-size agarose gels were prepared according to protocols.
According to protocols, 120 μl of 1kb Plus DNA ladder were prepared.

In the afternoon gel electrophoresis was performed.

As shown in figure, all clones were positive (apart from E13-1 run where it is possible to recognize only a band at approximately 2800 bp, corresponding to the linearized plasmid), so we cut and purified the bands of interest.

After gel extraction, digested DNA was quantified:

Part	DNA (ng/μl)
E9-2 (X-P)	5.9
E10-1 (X-P)	4.2
E11-1 (X-P)	5.2
E12-1 (X-P)	5.2
E16-1 (E-P)	6.4
E21-1 (E-P)	5.8
E22-2 (E-P)	6.5
E23-1 (E-P)	4.6
BBa_I13521	6.1
BBa_R0040 in BBa_J61002 (E-P)	2.1

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)
E24	BBa_R0040 (S-P)	1.5	E9-2 (X-P)	6.5	1	1
E25	BBa_R0040 (S-P)	1.5	E10-1 (X-P)	6.5	1	1
E26	BBa_R0040 (S-P)	1.5	E11-1 (X-P)	6.5	1	1
E27	<partinfo>BBa_R0040</partinfo> (S-P)	1.5	E12-1 (X-P)	6.5	1	1
E31	BBa_R0040 (E-P)	2.5	E16-1 (E-P)	5.5	1	1
E32	pSB4C5 (E-P)	2	E21-1 (E-P)	6	1	1
E33	pSB4C5 (E-P)	2	E22-2 (E-P)	6	1	1
E34	pSB4C5 (E-P)	6.5	E23-1 (E-P)	1.5	1	1
E35	pSB4C5 (E-P)	2	BBa_I13521 (E-P)	6.5	1	1
E36	pSB4C5 (E-P)	1	BBa_R0040 in BBa_J61002 (E-P)	7	1	1

Ligations were incubated ON at 16°C.

July, 19th

E13 was newly digested for the subsequent gel extraction.

Plasmid	Kind	DNA (μl)	H2O (μl)	Enzyme 1 (μl)	Enzyme 2 (μl)	Buffer H (μl)	Final Volume (μl)
E13	Insert	6	14.5	1 EcoRI	1 Pstl	2.5	25

After electrophoresis, E13 insert was succesfully identified and gel extracted (not shown). Meanwhile, sufficient amount of buffer 1 and buffer 2 for MGZ1 competent cell preparation protocol were prepared. E13 DNA was then quantified:

Plasmid	DNA (ng/μl)
E13 (X-P)	4.3

After E13 digestion (E-P), its ligation was performed in a final volume of 10 μl:

Ligation Name	Vector	Vector volume (μl)	Insert	Insert volume (μl)	Buffer (μl)	T4 Ligase (μl)
E28	pSB4C5 (E-P)	2	E13 (X-P)	6	1	1

Ligations were incubated ON at 16°C.

250 ml of M9 were prepared, according to protocols.

July, 20th

E24, E25, E26 and E27 were transformed in 100 μl of TOP10 competent cells according to protocols. Moreover E28, E31, E32, E33, E34, E35, E36 and BBa_B0032 (transformation efficiency positive control) were transformed in 100 μl of MGZ1 competent cells according to protocols. Plates were incubated ON at 37°C.

In the afternoon 500 ml of LB with chloramphenicol 12.5 were prepared.

July, 21th

All plates showed a lot of colonies, except for E28 and E31 (2 colonies).

A 26x mix was prepared in order to perform PCR on E24-1, E24-2, E25-1, E25-2, E26-1, E26-2, E27-1, E27-2, E28-1, E28-2, E31-1, E31-2, E32-1, E32-2, E33-1, E33-2, E34-1, E34-2, E35-1, E35-2, E36-1, E36-2:

H2O (μl)	Buffer 10x (μl)	MgCl2 (μl)	VF2 (BBa_G00100) μl	VR (BBa_G00101) μl	dNTPs (μl)	Taq polymerase (μl)
468	65	26	13	13	13	26

25 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 2 minutes (elongation)

35 cycles were performed.

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After the PCR, two medium size agarose gels were prepared in order to carry out DNA samples:

inserire immagini

All the band lengths were correct, except E31-1; 750 μl of E24-2, E25-1, E26-2, E27-2, E28-1, E31-1, E32-2, E33-2, E34-1 and E35-2 liquid cultures were used to prepare glycerol stocks.

July, 22th

E36 was transformed in 100 μl of TOP10 competent cells according to protocols. Plates were incubated ON at 37°C. E24-2, E25-1, E26-2, E27-2, E28-1, E31-1, E32-2, E33-2 and E34-1 plasmid purification was carried out, in order to prepare samples for sequencing.

Plasmid	DNA (ng/μl)
E24-2	76.8
E25-1	75.1
E26-2	69.4
E27-2	81.4
E28-1	18.1
E31-1	19.4
E32-2	21.0
E33-2	23.4
E34-1	25.8

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