Team:UNIPV-Pavia/Calendar/September/settimana4

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Latest revision as of 19:14, 27 October 2010

SEPTEMBER: WEEK 4

September, 20th

Screening PCR on 6 colonies picked from I55 was performed. Results were the following:

I55-1..6.

I55-3 seems to be ok.

Inoculum of I52, I53, I55, I56 and I57 in LB+Amp for MiniPrep of the following day

^top

September, 21st

Miniprep and Nanodrop quantification for:

I52 : 92,8 ng/ul
I53 : 118,5 ng/ul
I55 : 121,1 ng/ul
I56 : 159,6 ng/ul
I57 : 103 ng/ul

For I61 the DNA is already available quantified 147,1 ng/ul.

After 3 hour digestion was performed:

Culture	Kind	Final reaction volume (ul)	DNA (ul)	H20 (ul)	Enzyme 1 (ul)	Enzyme 2 (ul)	Buffer H (ul)
I52	Vector	25	10,8	9,7	1 EcoRI	1 XbaI	2,5
I53	Vector	25	8,5	12	1 EcoRI	1 XbaI	2,5
I55	Insert/Screening	25	14	6,5	1 EcoRI	1 SpeI	2,5
I56	Insert/screening	25	10	10,5	1 XbaI	1 PstI	2,5
I57	Vector	25	9,7	10,8	1 EcoRI	1 XbaI	2,5
I61	Vector	25	6,8	13,7	1 EcoRI	1 XbaI	2,5

Gel run/cut of samples

I52(E-X), I53(E-X), I55(E-S), I56(X-P), I57(E-X), I61(E-X).

As you can see only I52, I56, I57 and I61 run correctly (I55 was wrong) so gel extraction was performed on these samples:

I52 = 29,4 ng/ul
I56 = 15,4 ng/ul
I57 = 24,7 ng/ul
I61 = 30,2 ng/ul

We already had digested DNA so we could perform new ON ligations:

I62 = I39(S-P) + I59(X-P)
I63 = I38(E-S) + I61(E-X)
I64 = I58(E-S) + I52(E-X)
I65 = I39(S-P) + I56(X-P)
I66 = I60(E-S) + I57(E-X)

Tecan Test on self-inducible promoters was performed on prepared samples with a different protocol that is to bring the cultures to a known OD.

^top

September, 22nd

Trasformation of ligations I62, I63, I64, I65 and I66 into E.coli TOP10.

MiniPrep and quantification of:

MyYeast1 = 177,8 ng/ul
MyYeast2 = 98,9 ng/ul
MyYeast3 = 197,2 ng/ul

Screening digestion was performed as follows:

Culture	Kind	Final reaction volume (ul)	DNA (ul)	H20 (ul)	Enzyme 1 (ul)	Enzyme 2 (ul)	Buffer Tango (ul)
MyYeast1	Screening	25	1,2	19,3	1 EcoRI	1 NsiI	2,5
MyYeast2	Screening	25	2	18,5	1 EcoRI	1 NsiI	2,5
MyYeast3	Screening	25	1	19,5	1 EcoRI	1 NsiI	2,5

Gel run of samples was performed

MyYeast-1/2/3 (EcoRI-NsiI digest).

None of samples was positive.

Glycerol stocks of MyYeast1, MyYeast2, MyYeast3 was performed.

Inoculum in LB+Amp+Cm12,5 from glycerol stocks and colony for antibiotic resistance testing of I47_S1, I48_S1, I49_S1 and I13522.

^top

September, 23rd

Digestion of yesterday are repeted (because none of them were postitive) as follows:

Culture	Kind	Final reaction volume (ul)	DNA (ul)	H20 (ul)	Enzyme (ul)	Buffer H Roche 10X (ul)
MyYeast1	Screening	25	1,2	20,3	1 EcoRI	2,5
MyYeast2	Screening	25	2	19,5	1 EcoRI	2,5
MyYeast3	Screening	25	1	20,5	1 EcoRI	2,5

Culture	Kind	Final reaction volume (ul)	DNA (ul)	H20 (ul)	Enzyme (ul)	Buffer Red (ul)
MyYeast1	Screening	25	1,2	20,3	1 NsiI	2,5
MyYeast2	Screening	25	2	19,5	1 NsiI	2,5
MyYeast3	Screening	25	1	20,5	1 NsiI	2,5

Gel run of samples was performed

MyYeast-1/2/3 digest.

All samples seem to be positive!!

Inoculum for three colonies from I62, I63, I64, I65 agar plates into 5ml LB+Amp.

^top

September, 24th

Glycerol stocks was performed for

I62-1,2,3
I63-1,2,3
I64-1,2,3
I65-1,2,3
I66-1,2,3

MiniPrep and Nanodrop quantification :

I62-1 : 35,1 ng/ul
I62-2 : 27,8 ng/ul
I62-3 : 63,8 ng/ul
I63-1 : 195,3 ng/ul
I63-2 : 68,3 ng/ul
I63-3 : 148,7 ng/ul
I64-1 : 97,8 ng/ul
I64-2 : 171,8 ng/ul
I64-3 : 146,3 ng/ul
I65-1 : 69,7 ng/ul
I65-2 : 44 ng/ul
I65-3 : 294,3 ng/ul
I66-1 : 292,8 ng/ul
I66-2 : 198 ng/ul
I66-3 : 284,8 ng/ul

Digestion for screening :

I62-1,2,3 (E-P)
I63-1,2,3 (E-P)
I64-1,2,3 (E-P)
I65-1,2,3 (E-P)
I66-1,2,3 (E-P)

Gel run of samples was performed

Screening for I62/3/4/5/6 (E-P) digested.

As you can see I62-1/2/3 were all negative; I63-1/2/3, I64-1/2/3, I65-3, I66-1/2/3 were positive. So we made glycerol stocks for I63-1, I64-2, I65-3, I66-1 and stored them at -80°C. We stored their DNA in -20°C, too.

Dilution 1:50 of I47, I48, I49, GFP in LB+Amp+Cm12,5 and S1 in LB+Cm12,5.

All cultures were grow up to OD=0,1 of Nanodrop. They were then induced with HSL (lysis induction) 100nM

^top

September, 26th

Inoculum of <partinfo>BBa_K173000</partinfo>, I47, I48, I49, <partinfo>BBa_B0031</partinfo> into 3 ml LB+Amp for tomorrow TECAN test. Pick of six colonies from I66 agar plate (I66-A/B/C/D/E/F) and inoculum into 3 ml LB+Amp for screening thorough E-P digest. All colonies were let grow ON at 37°C, 220 rpm.

^top

@@ Line 37: / Line 37: @@
 ==September, 20th==
-Screening PCR on 6 colonies picked from I55 was performed. The results were the follow:
+Screening PCR on 6 colonies picked from I55 was performed. Results were the following:
-[[Image:UNIPV1020_09_10_I55.jpg|thumb|300px|center| I55.]]
+[[Image:UNIPV1020_09_10_I55.jpg|thumb|200px|center| I55-1..6.]]
-----
+I55-3 seems to be ok.
 Inoculum of I52, I53, I55, I56 and I57 in LB+Amp for MiniPrep of the following day
@@ Line 65: / Line 65: @@
 |    I53  ||  Vector|| 25 ||  8,5 ||  12   ||  1 EcoRI  || 1 XbaI   || 2,5
 |-
-|    I55  ||  Insert|| 25 ||  14 ||  6,5   ||  1 EcoRI  || 1 SpeI   || 2,5
+|    I55  ||  Insert/Screening|| 25 ||  14 ||  6,5   ||  1 EcoRI  || 1 SpeI   || 2,5
 |-
-|    I56  ||  Insert|| 25 ||  10 ||  10,5   ||  1 XbaI  || 1 PstI   || 2,5
+|    I56  ||  Insert/screening|| 25 ||  10 ||  10,5   ||  1 XbaI  || 1 PstI   || 2,5
 |-
 |    I57  ||  Vector|| 25 ||  9,7 ||  10,8   ||  1 EcoRI  || 1 XbaI   || 2,5
@@ Line 77: / Line 77: @@
 Gel run/cut of samples
-[[Image:UNIPV1021_09_10.jpg|thumb|300px|center]]
+[[Image:UNIPV1021_09_10.jpg|thumb|200px|center| I52(E-X), I53(E-X), I55(E-S), I56(X-P), I57(E-X), I61(E-X).]]
-As you can see only I52, I56, I57 and I61 run correctly so gel extraction was performed on these samples:
+As you can see only I52, I56, I57 and I61 run correctly (I55 was wrong) so gel extraction was performed on these samples:
 * I52 = 29,4 ng/ul
@@ Line 88: / Line 88: @@
 We already had digested DNA so we could perform new ON ligations:
-* I62 = I59(X-P) + I39(S-P)
+* I62 = I39(S-P) + I59(X-P)
-* I63 = I61(E-X) + I38(E-S)
+* I63 = I38(E-S) + I61(E-X)
-* I64 = I52(E-X) + I58(E-S)
+* I64 = I58(E-S) + I52(E-X)
 * I65 = I39(S-P) + I56(X-P)
-* I66 = I57(E-X) + I60(E-S)
+* I66 = I60(E-S) + I57(E-X)
 ----
-<font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test21settembre|Tecan Test]]</font> on self-inducible promoters was performed on prepared samples with a different protocol that is to bring the cultures to a known OD.
+Tecan Test on self-inducible promoters was performed on prepared samples with a different protocol that is to bring the cultures to a known OD.
 <div align="right"><small>[[#indice|^top]]</small></div>
@@ Line 100: / Line 100: @@
 ==September, 22nd==
-Trasformation of ligation of I62, I63, I64, I65 and I66 into E.coli TOP10.
+Trasformation of ligations I62, I63, I64, I65 and I66 into ''E.coli'' TOP10.
+----
 MiniPrep and quantification of:
@@ Line 120: / Line 123: @@
 |}
-Gel run of samples was performed(foto)
+Gel run of samples was performed
+[[Image:UNIPV10_22_09_10_MyYeast_screening.jpg|thumb|200px|center| MyYeast-1/2/3 (EcoRI-NsiI digest).]]
+None of samples was positive.
-Noone samples was positive
-----
 Glycerol stocks of MyYeast1, MyYeast2, MyYeast3 was performed.
@@ Line 136: / Line 137: @@
 ==September, 23rd==
-Digestion of yesterday are repeted (because noone of them was postitive) as follows:
+Digestion of yesterday are repeted (because none of them were postitive) as follows:
 {| border="1" align='center'
@@ Line 161: / Line 162: @@
 Gel run of samples was performed
-(foto)
+[[Image:UNIPV10_23_09_10_MyYeast_digest.jpg|thumb|200px|center|MyYeast-1/2/3 digest.]]
 All samples seem to be positive!!
@@ Line 167: / Line 169: @@
 ----
-Inoculum of I62, I63, I64, I65 and I66 of 3 colonies in LB+Amp 5ml.
+Inoculum for three colonies from I62, I63, I64, I65 agar plates into 5ml LB+Amp.
 <div align="right"><small>[[#indice|^top]]</small></div>
@@ Line 179: / Line 181: @@
 MiniPrep and Nanodrop quantification :
-* I62-1 : >30ng/ul
+* I62-1 : 35,1 ng/ul
-* I62-2 : >30ng/ul
+* I62-2 : 27,8 ng/ul
-* I62-3 : >30ng/ul
+* I62-3 : 63,8 ng/ul
-* I63-1 : >30ng/ul
+* I63-1 : 195,3 ng/ul
-* I63-2 : >30ng/ul
+* I63-2 : 68,3 ng/ul
-* I63-3 : >30ng/ul
+* I63-3 : 148,7 ng/ul
-* I64-1 : >30ng/ul
+* I64-1 : 97,8 ng/ul
-* I64-2 : >30ng/ul
+* I64-2 : 171,8 ng/ul
-* I64-3 : >30ng/ul
+* I64-3 : 146,3 ng/ul
-* I65-1 : >30ng/ul
+* I65-1 : 69,7 ng/ul
-* I65-2 : >30ng/ul
+* I65-2 : 44 ng/ul
-* I65-3 : >30ng/ul
+* I65-3 : 294,3 ng/ul
-* I66-1 : >30ng/ul
+* I66-1 : 292,8 ng/ul
-* I66-2 : >30ng/ul
+* I66-2 : 198 ng/ul
-* I66-3 : >30ng/ul
+* I66-3 : 284,8 ng/ul
-Screening digestion
+Digestion for screening :
+* I62-1,2,3 (E-P)
+* I63-1,2,3 (E-P)
+* I64-1,2,3 (E-P)
+* I65-1,2,3 (E-P)
+* I66-1,2,3 (E-P)
+Gel run of samples was performed
-<div align="right"><small>[[#indice|^top]]</small></div>
+[[Image:UNIPV10_24_09_10_screening_I62-3-4-5-6.jpg|thumb|200px|center| Screening for I62/3/4/5/6 (E-P) digested.]]
-==September, 25th==
+As you can see I62-1/2/3 were all negative; I63-1/2/3, I64-1/2/3, I65-3, I66-1/2/3 were positive. So we made glycerol stocks for I63-1, I64-2, I65-3, I66-1 and stored them at -80°C. We stored their DNA in -20°C, too.
+----
+Dilution 1:50 of I47, I48, I49, GFP in LB+Amp+Cm12,5 and S1 in LB+Cm12,5.
+All cultures were grow up to OD=0,1 of Nanodrop. They were then induced with HSL (lysis induction) 100nM
 <div align="right"><small>[[#indice|^top]]</small></div>
+==September, 26th==
+Inoculum of <partinfo>BBa_K173000</partinfo>, I47, I48, I49, <partinfo>BBa_B0031</partinfo> into 3 ml LB+Amp for tomorrow TECAN test. Pick of six colonies from I66 agar plate (I66-A/B/C/D/E/F) and inoculum into 3 ml LB+Amp for screening thorough E-P digest. All colonies were let grow ON at 37°C, 220 rpm.
+<div align="right"><small>[[#indice|^top]]</small></div>
 <!-- table previous next week -->
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