Team:UNIPV-Pavia/Calendar/July/settimana3

From 2010.igem.org

(Difference between revisions)
(July, 15th)
(July, 15th)
Line 266: Line 266:
|| I84C5-1 || I84C5-2 || I104C5-1 ||
|| I84C5-1 || I84C5-2 || I104C5-1 ||
|}
|}
 +
 +
Remaining cultures were re-filled with 5ml LB+abtibiotic and incubated (37°C, 220 rpm) for tomorrow screening.
I10-4C5-2 was grown also in LB+Amp, so it was thrown away!
I10-4C5-2 was grown also in LB+Amp, so it was thrown away!
Line 271: Line 273:
PBHR68 was grown, ok! :)
PBHR68 was grown, ok! :)
MiniPrep was performed for:
MiniPrep was performed for:
-
*PBHR68 -> 272 ng/ul
+
*PBHR68 (BioPlastic operon) -> 272 ng/ul
-
*<partinfo>BBa_J72007</partinfo> -> 41 ng/ul (not clean spectrum at 230nm)
+
*<partinfo>BBa_J72007</partinfo>(CRIM plasmid) -> 41 ng/ul (not clean spectrum at 230nm)
-
*<partinfo>BBa_J72013</partinfo> -> 16 ng/ul
+
*<partinfo>BBa_J72013</partinfo>(CRIM plasmid) -> 16 ng/ul
 +
 
 +
 
 +
Digestion of:
 +
 
 +
{| border="1" align='center'
 +
|  ''Culture''    ||  ''Kind''  ||  ''Final reaction volume (ul) '' ||  ''DNA (ul)''  ||  ''H20 (ul)''  ||  ''Enzyme 1''  ||  ''Enzyme 2'' ||  ''Buffer H''
 +
|-
 +
|  PBHR68  ||  Screening || 25 ||  1  ||  20,5  ||      1 EcoRI  || ---  || 2,5
 +
|-
 +
|  <partinfo>BBa_J72007</partinfo>  ||  Screening || 25 ||  5  ||  16,5  ||      1 XbaI  || ---  || 2,5
 +
|-
 +
|  <partinfo>BBa_J72013</partinfo>  ||  Screening || 25 || 10  ||  10,5  ||      1 SpeI  || 1 SacI  || 2,5
 +
|}
 +
 
 +
Digestions were incubated at 37°C for 3hours, then gel run/cut.
[[Image:UNIPV10_2_QC_CRIM_pBHR68.jpg|thumb|200px|center|Quality control for pBHR68 and CRIM plasmids]]
[[Image:UNIPV10_2_QC_CRIM_pBHR68.jpg|thumb|200px|center|Quality control for pBHR68 and CRIM plasmids]]
 +
 +
Quality control was ok for PBHR68, but no bands were observed for CRIM plasmids.
==July, 16th==
==July, 16th==

Revision as of 14:12, 29 July 2010

JULY: WEEK 3



July, 12th

Phasins PhaP1 and PhaP2 were sequenced, but none of them has a clear chromatogram, so we decided to sequence the PCR results.

Gel run/cut of phasins amplified via PCR

Phasins were gel run/cut and the samples were prepared to be sequenced.

Inoculum of

  • <partinfo>BBa_J23101</partinfo>
  • <partinfo>BBa_J23105</partinfo>
  • <partinfo>BBa_J23106</partinfo>

for tomorrow ligations.

July, 13th

LB+Amp was prepared and phasins sample were sent to be sequenced.

For cultures grown OverNight at 37°C, 220 rpm were MiniPrep was performed:

<partinfo>BBa_J23101</partinfo> 96,1 ng/ul
<partinfo>BBa_J23105</partinfo> 62,8 ng/ul
<partinfo>BBa_J23106</partinfo> 76,9 ng/ul

Other plasmids were retrieved from our freezer:

  • <partinfo>BBa_J23118</partinfo> (already digested SpeI-PstI)
  • <partinfo>BBa_J23110</partinfo> (already digested SpeI-PstI)
  • <partinfo>BBa_J23116</partinfo> (already digested SpeI-PstI)
  • I6-2 (already digested XbaI-PstI)
  • 4C5 (MiniPrep performed, it will be digested EcoRI-PstI)
  • I7-3 (MiniPrep performed, it will be digested EcoRI-PstI)
  • I8-5 (MiniPrep performed, it will be digested EcoRI-PstI)
  • I10-1 (MiniPrep performed, it will be digested EcoRI-PstI)
  • I3-1 (MiniPrep performed, it will be digested XbaI-PstI)


Digestion of:

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme 1 Enzyme 2 Buffer H
4C5 (x2) Vector 25 3,6 16,9 1 EcoRI 1 PstI 2,5
I7-3 Insert 25 14 6,5 1 EcoRI 1 PstI 2,5
I8-5 Insert 25 12,7 7,8 1 EcoRI 1 PstI 2,5
I10-1 Insert 25 15,3 5,2 1 EcoRI 1 PstI 2,5
I3-1 (x2) Vector 25 5,8 14,7 1 XbaI 1 PstI 2,5
<partinfo>BBa_J23105</partinfo> Insert 25 15,9 4,6 1SpeI 1 PstI 2,5
<partinfo>BBa_J23106</partinfo> Insert 25 13 7,5 1 SpeI 1 PstI 2,5
<partinfo>BBa_J23101</partinfo> Insert 25 10,4 10,1 1 SpeI 1 PstI 2,5

Digestions were incubated at 37°C for 3hours, then gel run/cut.

Gel was prepared for electrophoresis: 150ml TBE + 1,5 g Agarose + 3ul EtBr.

Ligations were all performed 1:5 (1ul vector + 5ul insert):

  • I11= <partinfo>BBa_J23101</partinfo> (S-P) + I6 (X-P)
  • I12= <partinfo>BBa_J23105</partinfo> (S-P) + I6 (X-P)
  • I13= <partinfo>BBa_J23106</partinfo> (S-P) + I6 (X-P)
  • I14= <partinfo>BBa_J23118</partinfo> (S-P) + I3 (X-P)
  • I15= <partinfo>BBa_J23110</partinfo> (S-P) + I3 (X-P)
  • I16= <partinfo>BBa_J23116</partinfo> (S-P) + I3 (X-P)
  • I17= <partinfo>BBa_J23101</partinfo> (S-P) + I3 (X-P)
  • I18= <partinfo>BBa_J23105</partinfo> (S-P) + I3 (X-P)
  • I19= <partinfo>BBa_J23106</partinfo> (S-P) + I3 (X-P)
  • I74C5= I7 (E-P) + 4C5 (E-P)
  • I84C5= I8 (E-P) + 4C5 (E-P)
  • I104C5= I10 (E-P) + 4C5 (E-P)

PhaP1 and PhaP2 samples were prepared for sequencing.

July, 14th

PBHR68 plate showed colonies!! We picked a colony and inoculated it in LB+Amp. This culture was grown at 37°C 220rpm for 6hours.

Trasformation of ligations in:

Ligation nameE. coli strain Resistance
  • I11= <partinfo>BBa_J23101</partinfo> (S-P) + I6 (X-P)

TOP10

Amp 100
  • I12= <partinfo>BBa_J23105</partinfo> (S-P) + I6 (X-P)

TOP10

Amp 100
  • I13= <partinfo>BBa_J23106</partinfo> (S-P) + I6 (X-P)

TOP10

Amp 100
  • I14= <partinfo>BBa_J23118</partinfo> (S-P) + I3 (X-P)

DH5alpha

Amp 100
  • I15= <partinfo>BBa_J23110</partinfo> (S-P) + I3 (X-P)

DH5alpha

Amp 100
  • I16= <partinfo>BBa_J23116</partinfo> (S-P) + I3 (X-P)

DH5alpha

Amp 100
  • I17= <partinfo>BBa_J23101</partinfo> (S-P) + I3 (X-P)

DH5alpha

Amp 100
  • I18= <partinfo>BBa_J23105</partinfo> (S-P) + I3 (X-P)

DH5alpha

Amp 100
  • I19= <partinfo>BBa_J23106</partinfo> (S-P) + I3 (X-P)

DH5alpha

Amp 100
  • I74C5= I7 (E-P) + 4C5 (E-P)

TOP10

Cm 12.5
  • I84C5= I8 (E-P) + 4C5 (E-P)

TOP10

Cm 12.5
  • I104C5= I10 (E-P) + 4C5 (E-P)

TOP10

Cm 12.5

PBHR68 plasmid was prepared, grown in LB+Amp and streaked on LB + Amp agar plate.

Quality control for HELPER and CRIM plasmids

Screening of <partinfo>BBa_J72007</partinfo> (CRIM), <partinfo>BBa_J72008</partinfo> (helper), pAH123 (helper) ans pCP20 (helper). MiniPrep was performed for these cultures (inoculum was performed yesterday).

<partinfo>BBa_J72007</partinfo> 45,2 ng/ul
<partinfo>BBa_J72008</partinfo> 78,0 ng/ul
pAH123 81,0 ng/ul
pCP20 45,2 ng/ul


Digestion of:

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme 1 Enzyme 2 Buffer H
<partinfo>BBa_J72007</partinfo> Screening 25 2 18,5 1 XbaI --- 2,5
<partinfo>BBa_J72008</partinfo> Screening 25 2 18,5 1 SpeI --- 2,5
pAH123 Screening 25 2 18,5 1 SpeI --- 2,5

These samples were digested at 37°C for 1 hour. Gel was loaded with digestions and with <partinfo>BBa_J72008</partinfo> and pCP20 not digested. Expected length for digestions was:

  • <partinfo>BBa_J72007</partinfo>: 1816 and 1351 bp
  • <partinfo>BBa_J72008</partinfo>: 3580 and 2755 bp (6335 not digested)
  • pAH123: 2755 and 2437
  • pCP20: 9400bp

Considered the results obtained, we decided to perform a further screening for <partinfo>BBa_J72007</partinfo>. Glycerol stock was prepared for PBHR68 and PBHR68 Backup, then falcon tube was re-filled with 5ml LB+Amp for further screening. Inoculum of <partinfo>BBa_J72007</partinfo> and <partinfo>BBa_J72013</partinfo> were performed in 5ml LB+Cm 34 (HC). Al falcon tubes were placed at 37°C 220 rpm.

Today we received our new enzymes!!!

  • MfeI
  • SbfI
  • NsiI

They were stored at -20°C in "Fermentas Enzymes" box.

July, 15th

We checked agar plates after transformation. All plates were ok, except for:

  • I11: only few colonies were observed
  • I10-4C5: only 2 colonies were observed
  • I15: only few colonies were observed


2 colonies were peaked from every plate and grown in 1ml LB + antibiotic at 37°C, 220rpm for 6 hours. (for colonies grown on LB+Cm, also 1ml LB+Amp was infected) After that, glycerol stocks were prepared for:

I11-1 I11-2 I12-1 I12-2
I13-1 I13-2 I14-1 I14-2
I15-1 I15-2 I16-1 I16-2
I17-1 I17-2 I18-1 I18-2
I19-1 I19-2 I74C5-1 I74C5-2
I84C5-1 I84C5-2 I104C5-1

Remaining cultures were re-filled with 5ml LB+abtibiotic and incubated (37°C, 220 rpm) for tomorrow screening.

I10-4C5-2 was grown also in LB+Amp, so it was thrown away!

PBHR68 was grown, ok! :) MiniPrep was performed for:

  • PBHR68 (BioPlastic operon) -> 272 ng/ul
  • <partinfo>BBa_J72007</partinfo>(CRIM plasmid) -> 41 ng/ul (not clean spectrum at 230nm)
  • <partinfo>BBa_J72013</partinfo>(CRIM plasmid) -> 16 ng/ul


Digestion of:

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme 1 Enzyme 2 Buffer H
PBHR68 Screening 25 1 20,5 1 EcoRI --- 2,5
<partinfo>BBa_J72007</partinfo> Screening 25 5 16,5 1 XbaI --- 2,5
<partinfo>BBa_J72013</partinfo> Screening 25 10 10,5 1 SpeI 1 SacI 2,5

Digestions were incubated at 37°C for 3hours, then gel run/cut.


Quality control for pBHR68 and CRIM plasmids

Quality control was ok for PBHR68, but no bands were observed for CRIM plasmids.

July, 16th

Screening ligations I11-I16
Screening ligations I17-I19, I7/I8/I10-4C5


Since it's impossible to isolate the CRIM plasmid <partinfo>BBa_J72007</partinfo> we created a pseudo CRIM plasmid to check that not pir strains aren't able to propagate it. We called it "THE RING" (RING) and we built it from our I5 (Cm resistance - <partinfo>BBa_P1004</partinfo> - + TT - <partinfo>BBa_B0015</partinfo> - + R6K ori - <partinfo>BBa_J61001</partinfo>) in <partinfo>pSB1A2</partinfo>:

  • Digestion of I5 XbaI-PstI
  • Gel extraction
  • Quantification with Nanodrop (15ng/ul)
  • Ligation of X and P sites each other (used 2ul of DNA)


Calendar

July

week 1

week 2

week 3

week 4

week 5