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Team:Technion/5 September 2012
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Ilya
- Repeated restriction of pCP backbone. Ran it on a gel. Got one big smear again.
- Prepared LB and BA media for next week.
- Cleaned the Fus2 and Fus2del inserts. Got good concentrations.
- Ran the inserts on a gel to see if they were destroyed, same as the backbone. The inserts are good. I figured out what was the problem. The glycerol stock of the pCP plasmid was in Stbl3 cells and not top10 cells (which we usually work with). Stbl3 are EndA+. We never did the optional wash step in the miniprep because because the top10 cells are EndA-. Apparently, EndA is the reason my backbone was destroyed.
- Put new starters of pCP. Rachel will miniprep them for me tomorrow (and she will do the optional wash step) and maybe advance to the other cloning steps.
Inbal
I got to say this week was the most productive week I had in a long LONG time! :]
Today I ran the restriction products of BBa_B0015 on 1% agarose gel for 30 minutes, I also ran uncut plasmid as control, the results were very good! the expected cut plasmid was 3300bp size. The uncut plasmid (pSB1AK3) ran up to 2200bp (I guess it because the circular plasmid was supercoiled), The linear plasmid (cut with XbaI) was approximately 3300bp! :] So I cut the 3300bp band and the concentration was 24.8ng/ul..
The purified cut plasmid was treated with cip enzyme for 1 hour at 37C. Then it was cleaned bu column cleaning kit, the concentration was 33ng/ul. After that, I ligated between the plasmid and T7* RNAP at 16C overnight.
Asaf
Because yesterday fusion PCR of the Riboswitch and the different polymerase genes didn't work,
I tried to find the right Tm that will work (yesterday I tried in Tm=69C).
I ran fusion PCR of the Riboswitch with K1F polymerase with gradient of temperatures.
This time I got bands in the proximity of the 3kb, but I'm not sure if it's the 3.2kb I was expecting.
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Hila
- Restriction to fragments 1C and 6C using PacI and SpeI.
- Clean the restriction reaction.
- Transformation of the ligated fragments (2C, 3C, 4C, 5C, 7C, 8C) into Top10-Rb bacteria.
Lior
Noa
Evgeni
- I've tried to grow starters for previous two days with no result, for some reason the cells are growing only on streak plates. I'll try new transformation attempt. Also I'll prepare more restricted plasmid, in case I'll have to do ligation again
- Putting 3 starters of cells with pPROLar in order to make more stock of pPROLar plasmid
Shahar
-Purification PCR product of T3 RNAP (the one with pSB1A2 primers) by PCR cleaning kit.
-Digest of T3 RNAP with XbaI and Spe for creating sticky ends.
-Clean the T3 RNAP after digestion by cleaning kit.
-This insert (T3 RNAP) is ready for cloning.
Rachel
-Purification PCR product of K1F RNAP (the one with pSB1A2 primers) by PCR cleaning kit.
-Digest of K1F RNAP with XbaI and Spe for creating sticky ends.
-Clean the K1F RNAP after digestion by cleaning kit. The final concentrations were: 19.8 ng/ul and 24.6 ng/ul.
-This insert (K1F RNAP) is ready for cloning.
