Team:UNIPV-Pavia/Notebook/Week4Jul

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== <html><font class="dayw_style">July, 22nd</font></html> ==
== <html><font class="dayw_style">July, 22nd</font></html> ==
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*We received the following stabs from iGEM HQ:
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*while F2620 will be shipped soon.
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*We transformed B1 and B2 overnight ligations in TOP10. Plates were incubated at 37°C overnight.
*We transformed B1 and B2 overnight ligations in TOP10. Plates were incubated at 37°C overnight.
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Revision as of 08:36, 23 July 2009

EthanolPVanimation.gif

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March 2009
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April 2009
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May 2009
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June 2009
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July 2009
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Week from July 20th, to July 26th, 2009

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July, 20th

  • Overnight digestion (20 ul reaction volume) for:
MRGENE1(X-P) MRGENE1-2(X-P) MRGENE1-3(X-P)
MRGENE2(X-P)(X2) MRGENE2-2(X-P) MRGENE2-3(X-P)
B0030(S-P)(X3)
  • NOTE: reactions which involve excision(X-P) were performed on 3 ug of DNA, while S-P digestions were performed with 1 ug of DNA. We decided to have 3 replicates to be sure to have an acceptable yield.
  • We decided to perform gel run/cut/purification for MRGENE2 and for B0030, while we decided to perform DNA precipitation with sodium acetate for MRGENE1.



  • We transformed about 2.5 pg of A14-4 in TOP10 in order to filter the wanted plasmids. We plated the transformed bacteria and incubated the plates at 37°C overnight.



  • We infected 5 ml of LB + Amp with 10 ul of A12-2 and A12-3 glycerol stocks. Tomorrow they will be miniprepped and sent to sequence!



Preparation of experiment with Tecan F200

  • We infected 5 ml of LB + Kan with a single colony taken from the native plate of B0015.
  • We incubated the inoculum for 6 hours (37°C, 220 rpm).



Experiment with Tecan F200

  • Description
  • Purpose:
  • Materials & Methods
  • Protocol
  • Results


Top

July, 21st

  • Gel run for:
    • MRGENE1(for check)
    • MRGENE2
    • MRGENE2-2
    • MRGENE2-3
    • B0030(X3)
  • We noticed unwanted bands in MRGENE1 run...So, we performed an analysis on Mr Gene plasmids and we found an unwanted PstI site in pMK-RQ that gives the noticed bands...
  • We decided to proceed to ligation and to perform a massive screening of the ligated inserts in the next days.
  • Gel purification for:
    • MRGENE2
    • MRGENE2-2
    • MRGENE2-3
    • B0030(X3)
  • DNA precipitation with sodium acetate for MRGENE1, MRGENE1-2, MRGENE1-3.
  • Results: after quantifications at Nanodrop, all the purified DNA samples had a good yield! let's proceed to ligation!
  • Ligation:
    • B1 = B0030(S-P) + MRGENE1(X-P)
    • B2 = B0030(S-P) + MRGENE2(X-P)



  • Miniprep for A12-2 and A12-3. We sent purified DNA to BMR Genomics for sequencing.



  • A14pg plate showed colonies! we picked 7 colonies and infected 5 ml of LB + Amp. We incubated the 7 inocula at 37°C, 220 rpm overnight.


  • We incubated the ligations at 16°C overnight.

Top

July, 22nd

  • We received the following stabs from iGEM HQ:


  • while F2620 will be shipped soon.


  • We transformed B1 and B2 overnight ligations in TOP10. Plates were incubated at 37°C overnight.



  • Glycerol stocks and miniprep for the 7 overnight cultures of A14pg.
  • Digestion E-P for the miniprepped DNA.
  • Gel run for these 7 samples.

Pv .jpg

  • Gel results: there was the expected ligated insert band in all the colonies, but some of them also have the non ligated insert (very small amount) or the same unexpected band (~1100 bp). None of these colonies was pure...We will discuss this result in the next days. If someone have suggestions about this problem, please e-mail lorenzo.pasotti@unipv.it . Thank you!

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July, 23rd

Top


July, 24th

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