Team:Paris Bettencourt/Notebook/VitaminB2

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13/07

• Received gBlocks RibA, RibD, RibE, RibT25 and RibT48 and amplification oligos from IDT.
Dilution in water and PCR amplification, using the following protocol:
• 1 μL gBlock (0.1 to 1ng)
• 1 μL forward primer (10 μM)
• 1 μL reverse primer (10 μM)
• 22 μL DNAse/RNAse free water
• 25 μL LifeTech MasterMix (2X)

RibA + o15.001 (GCGCCCGAAGACTTATGCAG) + o15.002(GGCCCCGCGCATATGAAG)
RibD + o15.003(CGCTATAGAAGACTTGAGAAGATCTG) + o15.004(GCGCGGCACCACATATGAAG)
RibE + o15.005(CGGCTATAGAAGACTTGCGC) + o15.006(CGCGCCGGCATATGAAGA)
RibT25 + o15.007(CCGCGTATAGAAGACTGCTAGA) + o15.008(CAGCAGCATATGAAGACAACCC)
RibT48 + o15.009(GCGGTATAGAAGACTGCTAGAGA) + o15.010(CAGCAGCATATGAAGACAACCC)

For each PCR reaction, a negative control without matrix DNA was prepared.



time (min)	temperature (°C)	function
3:00	98	melting
0:30	98	melting
0:30	52	annealing
1:00	72	extension
10:00	72	extension
forever	12	storage



Concentrations measured with a Nanodrop:

PCR purification using QIAGEN kit Add 5 volumes of resuspension buffer to 1 volume of PCR product in an 1.5mL microcentrifuge tube, mix by pipetting up and down Transfer in a centrifugation column Centrifuge 1 min at 14000 rpm Throw the filtration product Add 700μL of washing solution Centrifuge 1 min at 14000 rpm Throw the filtration product Add 500μL of washing solution Centrifuge 1 min at 14000 rpm Throw the filtration product Centrifuge 1 min at 14000 rpm Throw the filtration product Put the column in a sterile 1.5mL microcentrifuge tube Add 45μL of DNAse/RNAse free water on the membrane Wait 2 minutes Centrifuge 2min at 10000rpm Discard the column, DNA is saved in water

	[PCR product with gBlock] (ng/μL)	[PCR product without gBlock] (ng/μL)
RibA	3.5	3.4
RibD	6.4	3.3
RibE	5.0	3.7
RibT25	3.5	3.8
RibT48	8.7	3.3