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.
Inoculum of E. coli MGZ1 in 10 ml of LB for competentization.

July, 19th

According to protocols 200 ml of Buffer 1 and 100 ml of Buffer 2 were prepared for MGZ1 strain competentization.
E13-1 was newly digested.

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 gel electrophoresis, E13-1 insert was succesfully identified.

E13-1 digested DNA was gel-extracted and quantified:

Part	DNA (ng/μl)
E13-1 (E-P)	4.3

E28 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

Ligation was incubated ON at 16°C.

250 ml of M9 were prepared, according to protocols.
Competent MGZ1 cells were prepared according to protocols.

July, 20th

E24, E25, E26 and E27, E36 were transformed in 100 μl of TOP10 competent cells according to protocols. Moreover E28, E31, E32, E33, E34, E35 and BBa_B0032 (4 ng of DNA to test transformation efficiency) were transformed in 100 μl of MGZ1 competent cells according to protocols; a negative control was also plated in order to avoid the presence of contaminants. Plates were incubated ON at 37°C.
In the afternoon 33 LB + Cm 12.5 plates were prepared.

July, 21st

All plates showed a lot of colonies, except for E28, E31 (2 colonies) and E36; two colonies for each of them were picked, inoculated and screened by PCR. Positive control plate for MGZ1 showed few colonies due to poor transformation efficiency (new comptetent MGZ1 will be prepared!); no colonies grew on negative control plate.

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

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 30 seconds (elongation)

35 cycles were performed.

Two medium size agarose gels were prepared; in the afternoon gel electrophoresis was carried out:

All the band lengths were correct, except E31-2, E36-1 and E36-2 (E36 was plated on the wrong antibiotic!).
Glycerol stocks were prepared for E24-2, E25-1, E26-2, E27-2, E28-1, E31-1, E32-2, E33-2, E34-1 and E35-2.
Sequencing of E9-2, E10-1, E11-1, E12-1, E13-1, E16-1, E19-2, E20-2, E21-1, E22-2 and E23-1 were OK.

July, 22nd

E36 was transformed in 100 μl of TOP10 competent cells according to protocols. The plate was incubated at room temperature. E24-2, E25-1, E26-2, E27-2, E28-1, E31-1, E32-2, E33-2 and E34-1 plasmid purification was carried out:

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

E17-2, E18-2, E28-1, E31-1, E32-2, E33-2, E34-1, E35-2 purified DNA was sent for sequencing to BMR Genomics.
Other samples are ready for next week ligations.

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