Team:UNIPV-Pavia/Calendar/August/settimana3

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Latest revision as of 15:41, 7 October 2010

AUGUST: WEEK 3

August, 16th

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.

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August, 17th

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.

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August, 18th

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 (ul)	Enzyme 2 (ul)	Buffer H (ul)
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.

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August, 19th

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)

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August, 20th

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.

Colony PCR was performed for MyCRIM (9 colonies), 1 blank, 1 negative control (I5(X-S)), 1 positive control (MrGene CRIM from plate).

Screening PCR: MyCRIM (first 9 samples), ladder, blank, RING, CRIM from MrGene.

Tecan Test

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August, 21st

Plates with ligations I31..I41 were stored at +4°C. Colonies would have been screened ASAP.

Glycerol stocks and miniprep for MyCrim 1:9. Quantifications:

Culture	Quantification (ng/ul)
MyCRIM 1	59
MyCRIM 2	145
MyCRIM 3	108
MyCRIM 4	125
MyCRIM 5	65
MyCRIM 6	72
MyCRIM 7	97
MyCRIM 8	127
MyCRIM 9	151

Digestions were preformed to screen the direction of the fragment CAT-TT-ORI:

DNA	Buffer B	H2O	EcoRI	HindIII
2 ul	2.5 ul	18.5 ul	1 ul	1 ul

Screening digestions: MyCRIM (from 1 to 9).

The fragments length confirm us that all the samples has the CAT-TT-ORI fragment ligated in reversed way.

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@@ Line 50: / Line 50: @@
 ----
-Cultures were diluted (5ul in 2ml LB+antibiotic) for <font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test12|Tecan Test]]</font>:
+Cultures were diluted (5ul in 2ml LB+antibiotic) for <font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test17agosto|Tecan Test]]</font>:
 {| border='1' align='center'
@@ Line 72: / Line 72: @@
 ==August, 18th==
-Cultures were diluted (5ul in 2ml LB+antibiotic) for <font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test13|Tecan Test]]</font>:
+Cultures were diluted (5ul in 2ml LB+antibiotic) for <font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test18agosto|Tecan Test]]</font>:
 {| border='1' align='center'
@@ Line 190: / Line 190: @@
 ----
-<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test14|Tecan Test]]</font>
+Colony PCR was performed for MyCRIM (9 colonies), 1 blank, 1 negative control (I5(X-S)), 1 positive control (MrGene CRIM from plate).
+[[Image:UNIPV10_20_8_2010colonyPCRMyCREAM-AntarctPhosph9colonie_ladder_blank_RING_CRIMMrGene.jpg.jpg|thumb|300px|center|Screening PCR: MyCRIM (first 9 samples), ladder, blank, RING, CRIM from MrGene.]]
+<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test20agosto|Tecan Test]]</font>
 <div align="right"><small>[[#indice|^top]]</small></div>
 ==August, 21st==
 Plates with ligations I31..I41 were stored at +4°C. Colonies would have been screened ASAP.
+----
+Glycerol stocks and miniprep for MyCrim 1:9.
+Quantifications:
+{|align="center" border="1"
+|'''Culture'''||'''Quantification (ng/ul)'''
+|-
+|MyCRIM 1|| 59
+|-
+|MyCRIM 2|| 145
+|-
+|MyCRIM 3|| 108
+|-
+|MyCRIM 4|| 125
+|-
+|MyCRIM 5|| 65
+|-
+|MyCRIM 6|| 72
+|-
+|MyCRIM 7|| 97
+|-
+|MyCRIM 8|| 127
+|-
+|MyCRIM 9|| 151
+|-
+|}
+Digestions were preformed to screen the direction of the fragment CAT-TT-ORI:
+{|align="center" border="1"
+|'''DNA''' || '''Buffer B'''|| '''H2O''' || '''EcoRI''' || '''HindIII'''
+|-
+| 2 ul || 2.5 ul || 18.5 ul || 1 ul || 1 ul
+|-
+|}
+[[Image:UNIPV10_221agostoMyCRIMEco-Hind.jpg|thumb|300px|center|Screening digestions: MyCRIM (from 1 to 9).]]
+The fragments length confirm us that all the samples has the CAT-TT-ORI fragment ligated in reversed way.
 <div align="right"><small>[[#indice|^top]]</small></div>