Team:Technion/11 September 2012

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Revision as of 11:35, 26 September 2012

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Ilya

- Minipreped the picked colonies for testing the clones of Fus2 and Fus2del in pCP.
- Did a restriciton digest with BamHI-HF to test the colonies for the correct insert. 3/3 clones of Fus2del and 3/4 clones of Fus2 were positive. I picked one of each of the clones for plate reader tomorrow.
- Put starters for plate reader tomorrow.
- Tested ladders on a 0.6% agarose gel. Ran for an hour and 10 mins at 40V on a minigel. The 1Kb opened but the high range (HR) ladder didn't open. We will use a 0.4% agarose gel in the future attempts.
- Hila and I did the first attempt for gibson on the phage fragments. We attempted an assembly of fragments #4 (8080bp) and #5 (6150bp). The results are presented in the following gel pic:

As you can see in the attached file, only one of our three attempts today actually worked. The reaction conditions for the attempt that worked were: 100bp overlaps, ~150ng DNA of each of the templates (~0.025-0.03 pmoles), 5ul of Gibson mix and 5 ul of DNA fragments (10ul final volume). All reactions were incubated for 1 hr at 50 degrees as instructed by the manual.
I think that the 20 ul final volume reaction with the 100bp didn't work because there was too much DNA. The 400bp overlap might still work with additional exonuclease and in a 10ul volume with ~150ng of each fragment.

Inbal

-Miniprep for two samples out of three from the starters I made yesterday- because one of them has been contaminated. The conc's are: 146.5 and 142 ng/ul. I didn't know for sure if the insert (eT7) is within the plasmid (pSB1AK3) so I digested the plasmid with EcoRI. After running the products on a gel, comparison to the uncut plasmid, I got the desired band- 6kb approximately! The clone works!
- Shachar helped me to do a second transformation of native T3 to pSB1AK3 (2 transformations of 10ul and 6 ul from the ligation), we plated on 4 amp resistance plates (100ul and rest) and incubate them overnight at 37C.
- restriction of eT7+pSB1AK3 with XbaI, then I cleaned the products from buffers,etc.. and continue it to a cip reaction, after that I measured the concentration: 13.2 ng/ul. I ligated an inducible promoter (pTetO) attached to a reporter gene (m-cherry) to eT7+pSB1AK3, 37C for 1 hour.
- I transformed the ligation product into TOP10 component cells +control of cip.
- Starter for eT7+pSB1AK3 for cloning it to shipping plasmid.

Asaf

I cleaned the DNA from the extracted gel. I got a very low DNA concentration.

Hila

- Gel purification for F4 (400bp overlap), F4*(100bp overlap) and F5.
- Colony PCR for additional five colonies of fragments 4C, 5C and 7C. I found one positive colony for fragment 5.
- Transformation of ligated 1C and 6C to Top10-Rb bacteria.
- Starter for pBS1C3_MCS and for the positive colonies with the plasmid containing fragments 2C, 3C. 5C and 8C.
- Additional amplification for F4, F4* and F5.
- First Gibson assembly attempt. For more information – please read what Ilya wrote…

Lior

The trasformation worked, we have T7 promoter parts in a bacteria with PET system.
In order to test the system we used various concentrations of IPTG and checked absorbance for the ALP and xyIE part.
We also did coloby PCR for the positive control parts and made starters for the positive ones.

Noa

Evgeni

Shahar

after using the PCR products of N4 & T3 with pPROLAR primers as template - we needed to do another PCR to produce new N4 & T3 templates with pPROLAR prefix and sufix.
helped with colony PCR for additional five colonies of fragments 4C, 5C and 7C.
helped with transformation of ligated 1C and 6C to Top10-Rb bacteria.

Rachel

PCR for pTetO+m-cherry , after that i ran on gel the PCR products and got the band i expected for:800bp, then I cleaned the PCR products. The concentration was: 119 ng/ul.