Team:UNIPV-Pavia/Calendar/July/settimana3

July, 11th

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)
E1-2	Insert	18	2.5	1 XbaI	1 PstI	2.5	25
E2-2	Insert	18	2.5	1 XbaI	1 PstI	2.5	25
E3-1	Insert	18	2.5	1 XbaI	1 PstI	2.5	25
E4-2	Insert	18	2.5	1 XbaI	1 PstI	2.5	25
E5-2	Insert	18	2.5	1 XbaI	1 PstI	2.5	25
E6-1	Insert	11	9.5	1 XbaI	1 PstI	2.5	25
E7-2	Insert	18	2.5	1 XbaI	1 PstI	2.5	25
E8-3	Vector	20.5	0	1 SpeI	1 PstI	2.5	25
<partinfo>BBa_R0040</partinfo>	Vector	14.5	6	1 SpeI	1 PstI	2.5	25
<partinfo>BBa_C0261</partinfo>	Insert	18	2.5	1 XbaI	1 PstI	2.5	25
<partinfo>BBa_I13507</partinfo>	Insert	10	10.5	1 XbaI	1 PstI	2.5	25
<partinfo>BBa_B0034</partinfo>	Vector	10	10.5	1 SpeI	1 PstI	2.5	25

Reactions were incubated at 37°C for three hours while two medium-size agarose gels were prepared according to protocols.

In the afternoon gel electrophoresis was performed.

                
 link alle 2 img dei gel con ling wiki
                

As shown, in figure all clones were positive, so we cut and purified the bands of interest.

After gel extraction, cut DNA was quantified:

Plasmid	DNA (ng/μl)
E1 (X-P)	4.2
E2 (X-P)	3.8
E3 (X-P)	4.1
E4 (X-P)	3.7
E5 (X-P)	5.6
E6 (X-P)	7.8
E7 (X-P)	6.4
E8 (S-P)	3.4
<partinfo>BBa_C0261</partinfo> (X-P)	4.8
<partinfo>BBa_R0040</partinfo> (S-P)	7.9
<partinfo>BBa_B0034</partinfo> (S-P)	11.0
<partinfo>BBa_I13507</partinfo> (X-P)	6.8

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)
E9	<partinfo>BBa_B0030</partinfo> (S-P)	1	E1 (X-P)	7	1	1
E10	<partinfo>BBa_B0031</partinfo> (S-P)	1	E1 (X-P)	7	1	1
E11	<partinfo>BBa_B0032</partinfo> (S-P)	1	E1 (X-P)	7	1	1
E12	<partinfo>BBa_B0034</partinfo> (S-P)	1	E1 (X-P)	7	1	1
E13	<partinfo>BBa_R0040</partinfo> (S-P)	1.5	E2 (X-P)	6.5	1	1
E14	<partinfo>BBa_R0040</partinfo> (S-P)	1.5	E3 (X-P)	6.5	1	1
E15	<partinfo>BBa_R0040</partinfo> (S-P)	1.5	E4 (X-P)	6.5	1	1
E16	<partinfo>BBa_R0040</partinfo> (S-P)	2	<partinfo>BBa_C0261</partinfo> (X-P)	6	1	1
E17	E8 (S-P)	4.5	E5 (X-P)	3.5	1	1
E18	E8 (S-P)	5	E6 (X-P)	3	1	1
E19	E8 (S-P)	5	E7 (X-P)	3	1	1
E20	E8 (S-P)	5	<partinfo>BBa_I13507</partinfo> (X-P)	3	1	1
E21	<partinfo>BBa_R0040</partinfo> (S-P)	2	E5 (X-P)	6	1	1
E22	<partinfo>BBa_R0040</partinfo> (S-P)	2.5	E6 (X-P)	5.5	1	1
E23	<partinfo>BBa_R0040</partinfo> (S-P)	2	E7 (X-P)	6	1	1

Ligations were incubated ON at 16°C.

July, 12th

E9, E10, E11, E12, E13, E14, E15, E16, E17, E18, 19, E20, E21, E22 and E23 were transformed in 100 μl of TOP10 competent cells according to protocols. Plates were incubated ON at 37°C.

July, 13th

All plates showed a lot of colonies, except for E13, E16 (one colony),while for E14, E15 no colony was observed. Because of RFP, E21 was red, E22 and E23 a little bit less. Two colonies for E9, E10, E11, E12, E17, E18, E19, E20, E21, E22, E23 plates were picked. 500 ml of LB with Ampicillin were prepared. ???????????glicerolo stock. diciamo dei reinoculi????????????

July, 14th

A glycerol stock for E21-2, E23-2. E11-1 was prepared. Cultures grew overnight; plasmid purification and quantification were carried out:

Plasmid	DNA (ng/μl)
E9-1	114
E10-1	72
E11-1	78.5
E12-1	151.7
E13-1	133
E16-1	134.4
E17-1	27.8
E18-1	25.5

For E9-1, E9-2, E10-1, E10-2, E11-1, E11-2, E12-1, E12-2, E17-1, E18-2, E19-1, E19-2, E21-1, E20-2, E21-1, E21-2, E22-1, E22-2, E23-1, E23-2 PCR was performed. ??????????????????????????????????primer? da scrivere???????????????????????????????????????

In order to screen the DNA, a digestion for each ligation was performed.

DNA (μl)	H2O (μl)	Enzyme 1 (μl)	Enzyme 2 (μl)	Buffer H (μl)	Final Volume (μl)
3	18.5	0.5 EcoRI	0.5 PstI	2.5	25

Reactions were incubated at 37°C for three hours while two medium-size agarose gels were prepared according to protocols.

<partinfo>BBa_B0032</partinfo> were transformed in 100 μl of MGZ1 competent cells to test the competence according to protocols. Plates were incubated ON at 37°C.

In the afternoon gel electrophoresis was performed.

                
 link alle 2 img dei gel con ling wiki
                

As shown in figure, all clones were positive, except for E9-1,E17-2, E18-1, E18-2, E19-1 which didn' t show any DNA band. E9-2, E17-2, E18-2, E19-2, E20-2, E21-1, E22-2, E23-1 purification was carried out for sequencing.

Plasmid	DNA (ng/μl)
E9-2	66
E17-2	28
E18-2	26.3
E19-2	39.1
E20-2	22.1
E21-1	143.1
E22-2	70.4
E23-1	80.9
<partinfo>BBa_I13521</partinfo>	79

Cells harbouring E9-2, E10-1, E11-1, E12-1, E13, E16, E21-1, E22-2, E23-1 were inoculated in???? 5 ml???? LB + Amp.

July, 15th

All plates showed a lot of colonies, but the plate containing MGZ1 transformed with <partinfo>BBa_B0032</partinfo> also showed a lot of satellities.

In order to screen the DNA, digestionS for E17-1, E17-2, E18-1, E18-2 ligation were performed.

Ligation Name	DNA (μl)	H2O (μl)	Enzyme 1 (μl)	Enzyme 2 (μl)	Buffer H (μl)	Final Volume (μl)
E17-1	6	15.5	0.5 EcoRI	0.5 PstI	2.5	25
E17-2	6	15.5	0.5 EcoRI	0.5 PstI	2.5	25
E18-1	6	15.5	0.5 EcoRI	0.5 PstI	2.5	25
E18-2	6	15.5	0.5 EcoRI	0.5 PstI	2.5	25

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