Team:UNIPV-Pavia/Calendar/September/settimana3

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(Difference between revisions)
(September, 14th)
m (September, 14th)
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*I34: E-X (Vector)
*I34: E-X (Vector)
Gel run/cut of samples (I26 and I31 insert bands were very very soft)
Gel run/cut of samples (I26 and I31 insert bands were very very soft)
-
[[Image:UNIPV10_14_09_10_gel_run_cut_phasins_2step_final.jpg|thumb|300px|center| Gel run/cut for digested I26, I31 and I34.]]
+
[[Image:UNIPV10_14_09_10_gel_run_cut_phasins_2step_final.jpg|thumb|250px|center| Gel run/cut for digested I26, I31 and I34.]]
and gel extraction:
and gel extraction:
*I26 (X-P): 2,6 ng/ul
*I26 (X-P): 2,6 ng/ul

Revision as of 11:33, 16 September 2010


SEPTEMBER: WEEK 3



September, 13th

Unfortunately we discovered we tried to ligate I32 (E-S) to I37 (X-P) instead of I37 (E-X) ;-(

So this step will be repeated.

Trasformation of ligations

  • I52
  • I54
  • I55
  • I56
  • I57
  • I58

into E. coli DH5-alpha.

Inoculum of

  • I26
  • I31
  • I34

into 5 ml LB+Amp for ligations of the following day.


Inoculum from single colony of MG42 and MC43 in 5 ml LB+Cm12.5, than ON at 37°C.

September, 14th

Finally Mr.Gene sent us our YEAST: a little difficult to pick up the package but at the end we succeeded!


I52, I54, I55, I56, I57, I58 plates showed in general few colonies; I52 and I5X showed very few colonies (<=5). Very strange (mumble mumble...), they will be screened ASAP (stored at +4°C).

Miniprep and quanfification of:

  • I26: 69,7 ng/ul
  • I31: 89,5 ng/ul
  • I34: 147,6 ng/ul

3-hours digestion:

  • I26: XbaI-PstI (Insert)
  • I31: EcoRI-SpeI (Insert)
  • I34: E-X (Vector)

Gel run/cut of samples (I26 and I31 insert bands were very very soft)

Gel run/cut for digested I26, I31 and I34.

and gel extraction:

  • I26 (X-P): 2,6 ng/ul
  • I31 (E-S): 1,4 ng/ul
  • I34 (E-X): ng/ul

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

  • I59: I21 (E-S) + I34 (E-X)
  • I60: I20 (S-P) + I26 (X-P)
  • I61: I31 (E-S) + I37 (E-X)

and repeat

  • I53: I32 (E-S) + I37 (E-X)


Inoculum of I47, I48, I49 into 5 ml LB+Amp for TECAN test of the following day.


Screening PCR on 3 colonies picked from MC42 and MC43 respectively. Two method were used: our standard PCR picking a colony from the plate and using it for the PCR and a different protocol picking the colony and treating it with a 95°C 5 min step before the PCR.

The results were the follow:

MC42A/B/C, Cneg, blank, MC43A/B/C, Cneg, blank.

Negative controls were ok and maybe MC42_C and MC43_C were ok too but further investigations were necessary.

September, 15th

I47, I48, I49 cultures were diluted 1:100 into 5ml LB+Amp. In the afternoon all cultures were synchronized to O.D. 0,02 and Tecan Test was started to check GFP production by these BioBricks.


Transformation of I 53, I59, I60, I61 ligations into E. coli DH5-alpha. They were plated on LB+Amp agar plates and let grow ON at 37°C.



September, 16th

Screening through "colony PCR" for ligations:

  • I52
  • I53
  • I54
  • I55
  • I56
  • I57
  • I58
  • I59
  • I60
  • I61

For each plate we picked X colonies that were also inoculated into 1 ml LB+Amp in order to be ready to make glycerol stock of positive ones.

Agarose gel was prepared and samples loaded and run:

Gel run for ligations amplified through PCR.

As you can see ...


September, 17th

September, 18th