Team:UNIPV-Pavia/Calendar/August/settimana1

From 2010.igem.org

(Difference between revisions)
m (August, 2nd)
m (August, 2nd)
Line 55: Line 55:
|pAH123 ||rowspan="2"|  Amp 50 ug/ml ||rowspan="2"| 30°C
|pAH123 ||rowspan="2"|  Amp 50 ug/ml ||rowspan="2"| 30°C
|-
|-
-
|<partinfo>BBa_J72009</partinfo>
+
|<partinfo>BBa_J72008</partinfo>
|-
|-
|<partinfo>BBa_R0062</partinfo> || rowspan="2"| Amp 100 ug/ml ||rowspan="2"| 37°C
|<partinfo>BBa_R0062</partinfo> || rowspan="2"| Amp 100 ug/ml ||rowspan="2"| 37°C

Revision as of 19:06, 4 August 2010

AUGUST: WEEK 1



August, 2nd

Miniprep and quantification with Nanodrop of:

  • I20-1: 98,2 ng/ul
  • I20-2: 63,6 ng/ul
  • I20-3: 41,5 ng/ul
  • I21-1: 45 ng/ul
  • I21-2: 45 ng/ul
  • I21-3: 54 ng/ul

These samples were prepared and sent (400ng) to BMR Genomics for sequencing.


The following parts were resuspended from iGEM 2010 Distribution Kit:

  • <partinfo>BBa_R0062</partinfo> (Plate 1, Well 6O)
  • <partinfo>BBa_K081009</partinfo> (Plate 2, Well 10N)

both in vector <partinfo>pSB1A2</partinfo>.

Transformation (1ul) of the following parts (resuspended/already miniprepped):

Part Strain Culture name
pAH123 MC1061 MC123
MG1655 MG123
<partinfo>BBa_J72008</partinfo> MC1061 MC008
MG1655 MG008
<partinfo>BBa_R0062</partinfo> DH5-alpha
<partinfo>BBa_K081009</partinfo>

Transformed cells were plated on proper LB+Amp agar plates and grown ON at right temperature:

Part Plate resistance Temperature
pAH123 Amp 50 ug/ml 30°C
<partinfo>BBa_J72008</partinfo>
<partinfo>BBa_R0062</partinfo> Amp 100 ug/ml 37°C
<partinfo>BBa_K081009</partinfo>

August, 3rd

Check for plates grown ON: all plates showed colonies.

<partinfo>BBa_R0062</partinfo> plate
<partinfo>BBa_K081009</partinfo> plate

A single colony was picked from <partinfo>BBa_R0062</partinfo> and <partinfo>BBa_K081009</partinfo> plates and inoculated in 1 ml LB+Amp 100 ug/ml and incubated 37°C, 220 rmp for glycerol stock. Cultures left were refilled to 5 ml of proper medium and incubated ON at 37°C, 220 rpm for further screening.

Since plates left showed small colonies they were let grow until late afternoon; than some colonies were picked from each plate and inoculated into a 5 ml LB+Amp 50 ug/ml falcon. Falcon tubes were incubated and shaken ON at 30°C.

MC1061 transformed with pAH123
MC1061 transformed with <partinfo>BBa_J72008</partinfo>
MG1655 transformed with pAH123
MG1655 transformed with <partinfo>BBa_J72008</partinfo>

PCR from the following colonies (this is a test for the efficiency of our primers synthesized to check attPhi80 E. coli genomic integration and it will be our negative control for future screenings):

  • MC1061-1
  • MC1061-2
  • MG1655-1
  • MG1655-2
  • Blank (Nothing)

Gel run of amplified DNA showed for every sample the expected distance between primers of a strain with nothing integrated in attPhi80 site (~XXX bp), but unfortunately we forgot to take a picture of the gel ;(

August, 4th

Glycerol stocks for MC1061 and MG1655 strains transformed with pAH123 or <partinfo>BBa_J72008</partinfo> helper plasmids.

2 ul of MC1061 bacteria were transferred into 5 ml LB+Amp 50 ug/ml and grown and shaken at 30°C over-day and over-night for re-competentization of the following day.

200 ul of MG1655 cultures were transferred into 100 ml LB+Amp 50 ug/ml and grown and shaken at 30°C for re-competentization of today.

All cultures were miniprepped to check again the presence of helper plasmids.

Miniprep gave the following results:

  • MC1061 with pAH123: 20,5 ng/ul
  • MC1061 with <partinfo>BBa_J72008</partinfo>: 20,5 ng/ul
  • MG1655 with pAH123: 20,5 ng/ul
  • MG1655 with <partinfo>BBa_J72008</partinfo>: 20,5 ng/ul

Samples were digested with SpeI ()it cuts twice for 3 hours and gel run:

  • pAH123 digested: 3376 and 939 bp
  • <partinfo>BBa_J72008</partinfo> digested: 4519 and 939 bp
File:UNIPV10 MG 1655 MC1061 helper screening.jpg
MG1655 and MC1061 transformed with pAH123 and <partinfo>BBa_J72008</partinfo> screening

Unfortunately we got a bad gel run so this time we decided to pick two single colonies from each of the plates made on August, 3rd and to inoculate them into 5 ml LB+Amp 50 ug/ml. A total of eight falcon tubes was incubated ON at 30°C, 220 rpm.

We plan to screen them (check the presence of helper plasmids) and to re-competentize only the positive ones.

August, 5th

August, 6th

August, 7th

August, 8th

Calendar

August

week 1

week 2

week 3

week 4

week 5