Team:UNIPV-Pavia/Calendar/August/settimana4

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AUGUST: WEEK 4

August, 23rd

Resuspension from iGEM 2010 Distribution Kit of <partinfo>BBa_J13002</partinfo> (Plate 1, Well 13B) and <partinfo>BBa_I13522</partinfo> (Plate 2, Well 8A).

Transformation (1 ul) of <partinfo>BBa_J13002</partinfo>, <partinfo>BBa_I13522</partinfo>, INTEIN, CREAM (Mr.Gene) into 100 ul of E. coli TOP10 home-made competent cells.

Transformation of 15-4C5 (1 ul) into in re-competentized E. coli TOP10 carrying <partinfo>BBa_T9002</partinfo>.

Transfomants were plated on LB+Amp 100 ug/ml agar plates, the last one on LB+Amp100+Cm12,5 agar plates and were let grow ON at 37°C.

August, 24th

All plates showed colonies.

Pick of <partinfo>BBa_J13002</partinfo>, <partinfo>BBa_I13522</partinfo>, INTEIN, CREAM, co-trasformed <partinfo>BBa_T9002</partinfo> and inoculum into 1 ml of LB+Amp, LB+Kan, LB+Cm, respectively, to make glycerol stocks. Falcons were than refilled for the minipreps of the following day.

Inoculum of I5, I26, <partinfo>BBa_K105012</partinfo>, <partinfo>BBa_B0034</partinfo>, I20M-1, I20A-1, I21M-3, I21-4, I21A-7, I0 for minipreps of the following day.

Cultures were diluted (5ul in 2ml LB+antibiotic) for TECAN test:

I7-A	I8-5 D	I9-1
I10-B	I12-2	A2
<partinfo>BBa_B0032</partinfo>	I144C5-T9002	I164C5-T9002
I174C5-T9002	I184C5-T9002	I194C5-T9002
ENTERO4C5-T9002

Glycerol stock was prepared for ENTERO4C5-T9002.

August, 25th

Cultures were diluted (5ul in 2ml LB+antibiotic) for TECAN test:

I7-A	I8-5 D	I9-1
I10-B	I12-2	A2
<partinfo>BBa_B0032</partinfo>	I144C5-T9002	I154C5-T9002
I164C5-T9002	I174C5-T9002	I184C5-T9002
I194C5-T9002	ENTERO4C5-T9002

Glycerol stock was prepared for I154C5-T9002.

The following cultures were miniprepped and quantified with Nanodrop:

I26: 67,5 ng/ul
<partinfo>BBa_K105012</partinfo>: 60,9 ng/ul
<partinfo>BBa_B0034</partinfo>: 64,5 ng/ul
<partinfo>BBa_F2620</partinfo>: 91,8 ng/ul
I20M-1: 191,8 ng/ul
I20A-1: 176,2 ng/ul
I21M-3: 156,2 ng/ul
I21-4: 76,3 ng/ul
I0: 121,2 ng/ul
INTEIN: 143,9 ng/ul
<partinfo>J13002</partinfo>: 31,9 ng/ul

and than digested as follows for four hours:

Culture	Kind	Final reaction volume (ul)	DNA (ul)	H20 (ul)	Enzyme 1	Enzyme 2	Buffer H
I26	Insert	25	21,5	0	0,5 EcoRI	0,5 SpeI	2,5
<partinfo>BBa_K105012</partinfo>	Vector	25	16,5	5	0,5 E	0,5 S	2,5
<partinfo>BB_B0034</partinfo>	Vector	25	15,5	6	0,5 E	0,5 S	2,5
<partinfo>BB_F2620</partinfo>	Insert	25	16,5	5	0,5 E	0,5 S	2,5
I20M-1	Insert/Screening	25	8	13,5	0,5 E	0,5 S	2,5
I20M-1	Insert/Screening	25	8	13,5	0,5 XbaI	0,5 PstI	2,5
I20A-1	Insert/Screening	25	13	8,5	0,5 E	0,5 S	2,5
I20A-1	Insert/Screening	25	13	8,5	0,5 X	0,5 P	2,5
I21-4	Insert/Screening	25	13	8,5	0,5 E	0,5 S	2,5
I21-4	Vector	25	13,1	8,4	0,5 E	0,5 X	2,5
I0	Vector	25	8,2	13,3	0,5 E	0,5 XbaI	2,5
INTEIN	Insert	25	10,5	11	0,5 E	0,5 S	2,5
<partinfo>BB_J13002</partinfo>	Vector	25	21,5	0	0,5 S	0,5 P	2,5

Two medium gel were prepared and samples were loaded and gel run/cut.

Vectors digested and gel run/cut

Inserts digested and gel run/cut

All samples were correctly digested and run; both I20M-1 and I20A-1 are positive, so we decided to use I20M-1; this new screening for I21-4 showed again that it's correct.

Gel extraction gave these results:

I0 (E-X): 20,8 ng/ul
<partinfo>BB_F2620</partinfo> (E-S): 13,5 ng/ul
INTEIN (E-S): 6,5 ng/ul
<partinfo>BB_B0034</partinfo> (E-X): 23,2 ng/ul
I20M-1 (E-S): 7,4 ng/ul
<partinfo>BB_J13002</partinfo> (S-P): 12,5 ng/ul
I21-4 (E-S): 7,2 ng/ul
I21-4 (E-X): 24,7 ng/ul
<partinfo>BBa_K105012</partinfo> (E-X): 23,1 ng/ul
I26 (E-S): 3,4 ng/ul
I20M-1 (X-P): 6,5 ng/ul

They were stored ON at -20°C.

August, 26th

Extra E-X digestion (four hours) of previously miniprepped INTEIN

Culture	Kind	H2O	DNA	Enzyme1	Enzyme2	Buffer H
INTEIN	Vector	13,5 ul	7 ul	1 ul EcoRI	1 ul XbaI	2,5 ul

Sample was loaded into a small agarose gel, run and cut.

After gel estraction was quantified with Nanodrop: 19,6 ng/ul.

Finally were performed the following ligations:

I31: I20M-1 (E-S) + <partinfo>BBa_K105012</partinfo> (E-X)
I32: I20M-1 (E-S) + I21-4 (E-X)
I33: I20M-1 (E-S) + I0 (E-X)
I34: I21-4 (E-S) + I0 (E-X)
I35: I21-4 (E-S) + <partinfo>BBa_K105012</partinfo> (E-X)
I36: I21-4 (E-S) + I21-4 (E-X)
I37: INTEIN (E-S) + I0 (E-X)
I38: <partinfo>BBa_F2620</partinfo> + <partinfo>BBa_B0034</partinfo>
I39: <partinfo>BBa_J13002</partinfo> + I20M-1 (X-P)
I40: I26 (E-S) + INTEIN (E-X)
I41: I26 (E-S) + I0 (E-X)

August, 27th

Ligations of previous day were transformed (1 ul) into 100 ul of home-made competent cells 'E. coli DH5-alpha. They were than plated on LB+Amp agar plates and incubated ON at 37°C.

August, 28st

Plates with ligations I31..I41 were stored at +4°C. Colonies will be screened ASAP.