(Difference between revisions)

Week from July 20th, to July 26th, 2009

Previous Week

Next Week

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

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.

July, 22nd

• 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.

• 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!

July, 23rd

July, 24th

---

Previous Week

Next Week