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Team:Brown/Notebook/June18
From 2010.igem.org
Friday, June 18 2010
After meeting with Gary in the morning and trying to figure out Will’s primer design, we performed the restriction digest and PCR.
Double Digestion of pAHtathis2 (from now on designated as pWill1)
- 8 µl dH2O
- 2 µl 10X NEB Buffer 4 (checked on Double Digest finder)
- 8 µl DNA (pAHtatHis2)
- 1 µl NdeI
- 1 µl XhoI
-- 20 µl total volume
37°C for 1’; Begin at 3:15 PM; took out at 4:15 PM and placed on ice.
Cast a 1% agarose gel
- 0.6114g agarose in ~61 mL of 1X TAE
Ran gel for digest products (20 µl volume + 1 µl loading dye)
- Lane 1: ladder (10 µl)
- Lane 2: 10 µl of digest product
- Lane 3: 10 µl of digest product
- Lane 4: blank
- Lane 5: blank
Soaked in EtBr for 10 minutes
Expected:
- pJExpress backbone = just under 4kb
- Will1 Construct = ~850 bp
- pWill1 undigested = ~4.8 kb
Results: We failed to detect DNA bands from looking at it through the gel box (UV light illuminator). The ladder was faintly visible, but for the amount we loaded, we expected to see more visible bands. Perhaps we should amplify Will’s DNA construct before trying again by cloning it into E. coli.
Overview of PCR addition of BamHI and NcoI to pWill1’s ORF
Will’s Primers: Overlap with 5’ end of pWill1 construct ORF
- A 5’ – ATA GGA TCC TAC GGT CGC AAG – 3’
BamHI, XhoII
- B 5’ – AAC CAT GGA CAG TAC TGC CAG – 3’
NcoI Reverse complement overlaps with 3’ end of Will’s construct ORF
