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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 transformed B1 and B2 overnight ligations in TOP10. Plates were incubated at 37°C overnight. | ||
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| + | *Glycerol stocks and miniprep for the 7 overnight cultures of A14pg. | ||
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| + | *Digestion E-P for the miniprepped DNA. | ||
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| + | *Gel run for these 7 samples. | ||
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== <html><font class="dayw_style">July, 23rd</font></html> == | == <html><font class="dayw_style">July, 23rd</font></html> == | ||
Revision as of 02:49, 23 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
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.
July, 23rd
July, 24th
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