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Team:Technion/9 August 2012
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Today I amplified by PCR the SP6 gene (length: 2700 bp, including primers)again from the pACSP6R plasmid using 2 separate buffers for 2 reactions: GC and HF buffers- to see which buffer works the best for amplifying the gene. | Today I amplified by PCR the SP6 gene (length: 2700 bp, including primers)again from the pACSP6R plasmid using 2 separate buffers for 2 reactions: GC and HF buffers- to see which buffer works the best for amplifying the gene. | ||
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| - | After that, I measured the concentration of each reaction: GC- | + | After that, I measured the concentration of each reaction: GC- 309.5ng/ul , HF-509 ng/ul <br> |
After PCR, I ran the products in agarose gel and sliced the product band originated from the GC reaction (2700bp). I will mention that I didn't see band at 2700 bp in the HF reaction, instead I saw smear at the bottom of the gel (probably primers) . | After PCR, I ran the products in agarose gel and sliced the product band originated from the GC reaction (2700bp). I will mention that I didn't see band at 2700 bp in the HF reaction, instead I saw smear at the bottom of the gel (probably primers) . | ||
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Latest revision as of 12:21, 13 August 2012
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Ilya
- Sent BBa F2620 and BBa J23119 to sequencing.
- Analized the plate reader results for tac+RS+mCherry. Came to the conclusion that the main issue is probably the high copy number of the plasmid. Consulted with members of the lab, Sarah told me I need a true positive control, without the riboswitch. The plan now is to clone the construct into a low copy plasmid, generate a positive control plasmid and repeat the plate reader assay. I will also continue the cloning of I0462 into the R0062+RS+mCherry construct.
- Helped the other groups plan their primers and cloning steps.
- Fetched the spectinomycin from a lab in tne biology faculty and sent Orna the catalog number.
- Started planing a plasmid with an MCS.
Inbal
Today I amplified by PCR the SP6 gene (length: 2700 bp, including primers)again from the pACSP6R plasmid using 2 separate buffers for 2 reactions: GC and HF buffers- to see which buffer works the best for amplifying the gene.
After that, I measured the concentration of each reaction: GC- 309.5ng/ul , HF-509 ng/ul
After PCR, I ran the products in agarose gel and sliced the product band originated from the GC reaction (2700bp). I will mention that I didn't see band at 2700 bp in the HF reaction, instead I saw smear at the bottom of the gel (probably primers) .
The sliced band is stored at 4 Celsius degrees.
In addition, Hila and me prepared LB+CM+spectinomycin plates.
Asaf
Hila
Lior
Cip- me and Noa took 500 ul from the starter and made a glycerol stock for the part and sent the part for sequencing.
xyIE- we minipreped the plasmid with the part. We got 140.5 ng/ul. 260/280= 1.938. Moreover we made a glycerol stock for the part.
