Team:UNIPV-Pavia/Calendar/July/settimana5

July, 25th

Red colonies were observed from E36 plate, suggesting that the transformation was successful. A colony was picked form the plate.

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)
E24-2	Insert	13	7.5	1 XbaI	1 PstI	2.5	25
E25-1	Insert	13.5	7	1 XbaI	1 PstI	2.5	25
E26-2	Insert	14.5	6	1 XbaI	1 PstI	2.5	25
E27-2	Insert	12.5	8	1 XbaI	1 PstI	2.5	25

Reactions were incubated at 37°C for three hours while a small size agarose gel was prepared according to protocols.
In the afternoon gel electrophoresis was performed:

After gel extraction, digested DNA was quantified:

Part	DNA (ng/μl)
E24-2 (E-P)	7.4
E25-1 (E-P)	8.4
E26-2 (E-P)	3.2
E27-2 (E-P)	6.3

Ligations were performed:

Ligation Name	Vector	Vector volume (μl)	Insert	Insert volume (μl)	Buffer (μl)	T4 Ligase (μl)
E37	pSB4C5 (E-P)	2	E24-2 (E-P)	6	1	1
E38	pSB4C5 (E-P)	2.5	E25-1 (E-P)	5.5	1	1
E39	pSB4C5 (E-P)	1	E26-2 (E-P)	7	1	1
E40	pSB4C5 (E-P)	2	E27-2 (E-P)	6	1	1

Inoculum of E17-2, E18-2, E19-2 or E20-2 to amplify DNA and transfer it into MGZ1; refill for E36 liquid culture.
Glycerol stock fpr E36 was prepared
250 ml of LB + Cm 12.5 were prepared.

July, 26th

Plasmids containing E17-2, E18-2, E19-2, E20-2 and E36 were extracted with MiniPrep kit from ON saturated cultures; purified DNA was quantified with NanoDrop Spectrophotometer:

Plasmid	DNA (ng/μl)
E17-2	20.3
E18-2	18.6
E19-2	17.3
E20-2	13.2
E36	17.8

Digestion of E36 was performed for ligations:

Plasmid	Kind	DNA (μl)	H2O (μl)	Enzyme 1 (μl)	Enzyme 2 (μl)	Buffer H (μl)	Final Volume (μl)
E36	Vector	20.5	0	1 Spel	1 Pstl	2.5	25

The sample was incubated at 37°C for three hours while a small-size agarose gel was prepared; in the afteroon gel electrophoresis was carried out:

After gel extraction, digested DNA was quantified:

Part	DNA (ng/μl)
E36 (S-P)	3.5

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)
E41	E36 (S-P)	4	E3-1 (X-P)	4	1	1
E42	E36 (S-P)	3.5	E4-2 (X-P)	4.5	1	1

Ligations were incubated at 16°C ON.

July, 27th

E37, E38, E39, E40, E41, E42, E17, E18, E19 and E20 were transformed in 100 μl of MGZ1 competent cells according to protocols. Moreover 4ng of purified BBa_B0032 DNA were transformed in MGZ1 in order to test the transformation efficiency. Plates were incubated ON at 37°C.
BMR Genomics sent results of parts sequencing; E17-2, E18-2, E31-1, E32-2, E33-2, E34-1, E35-2 were OK while E28-1 sequence was wrong.

July, 28th

Colonies grew in every plate. In fact E17-2, E18-2, E19-2 and E20-2, E37, E38, E39 and E40 showed grown colonies. We picked two colonies for each plate and inoculate them in 5 ml of LB + Cm 12.5. As regards to E41 and E42 parts, we resolved to let them still grow; in the late morning we picked and inoculate two colonies for both plates.
Plate with BBa_B0032 showed 9 colonies; the corresponding efficiency results in 2250 colonies/μg of DNA (very low, we need to prepare new competent MGZ1).

July, 29th

Plasmid purification was carried out on over-night grown cultures (E41-1 did not grow!):

Plasmid	DNA (ng/μl)
E37-1	16.6
E37-2	16.9
E38-1	28.4
E38-2	15.0
E39-1	15.0
E39-2	14.5
E40-1	17.1
E40-2	38.9
E41-2	13.7
E42-1	22.7
E42-2	28

A 12x mix was prepared for screening digestions:

H2O (μl)	Enzyme 1 (μl)	Enzyme 2 (μl)	Buffer H (μl)
126	12 EcoRI	12 PstI	30

For each digestion 10 μl of purified DNA were used. Meanwhile a medium size gel was prepared.
In the afternoon gel electrophoresis was carried out:

All clones were positive except for E42-2 and E41-2.
Glycerol stocks were prepared for E37-2, E38-1, E39-1, E40-2 and E42-1.
Ligations of consitutive promoter BBa_J23101 with different RBS in pSB4C5 were performed:

Ligation Name	Vector	Vector volume (μl)	Insert	Insert volume (μl)	Buffer (μl)	T4 Ligase (μl)
BBa_J23101-31	BBa_J23101 in pSB4C5 (S-P)	4	E6 (X-P)	4	1	1
BBa_J23101-E5	BBa_J23101 in pSB4C5 (S-P)	4	E5 (X-P)	4	1	1
BBa_J23101-E7	BBa_J23101 in pSB4C5 (S-P)	4	E7 (X-P)	4	1	1

July, 30th

E28 and E41 were again transformed in 200 μl of MGZ1 competent cells.
J101-E5, J101-31 and J101-E7 ligations were transformed in 100 μl of TOP10 competent cells.

July, 31st

All plates showed colonies. One colony was picked from each of J101-E5, J101-31 and J101-E7 plates, inoculated in LB + Cm 12.5 and grown.

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