IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-10-20

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Gel extraction of 010 and 001

Gel extracted using the 5 mL tubes with caps. Heat block that contained larger slots was used so that the 5 mL tubes could fit. In retrospect, the Gel purification should have used more than 2 Qiagen spin columns - hence one reason for the low amounts of DNA.

pcr purification of 010 and 001

PCR purified - good numbers for 010, not so good for 001 (~9 ng/uL). In 30uL EB. Valuable:D

Ligation

For ligation, we use the Roche 5min quick ligase kit. Vial 3 is generic T4 DNA ligase.

Regarding the contents of the buffer 2: Called, they said that the DNA dilution buffer was proprietary but one should use 2ul of the 5X stock + 8uL DNA to add to 10uL. Checked the pH of the Dilution Buffer, and it either is set around 7 or actually is not a pH buffer at all.

Regarding buffer 1: It contains ATP, so try not to freeze/thaw the stock.

It is still unknown if a 1:3 volume ratio or a 1:3 molar ratio is ideal for ligations. I personally use the latter and would recommend that over the former. 1:1 may be good for small inserts, big vectors; we havent tested it though so we're not sure.

Ligation of J36001 and J04500:

 2uL 5x stock (buffer #2)
 1uL Vector DNA (the thing cut with \sp)
 7uL Insert DNA (the thing cut with \xp) 
 10ul Ligation buffer (buffer #3)
 1ul ligase (buffer #1)

Ligation of J36010 and J36012:

 2uL 5x stock (buffer #2)
 1uL Vector DNA (the thing cut with \sp)
 7uL Insert DNA (the thing cut with \xp) 
 10ul Ligation buffer (buffer #3)
 1ul ligase (buffer #1)
  • USE the 5x DNA dilution buffer; do not skimp on it!
  • Let sit at Room Temperature for 30min to 1h or longer.


USE 2uL of the ligation product in the transformation. NOT the whole thing! 20 uL of competent cells, 5 trials per transformation, ranging from 1uL to 5uL of ligation product. also transformed 1uL of terminator B0015. Carb plates, the usual chemically competent cells transformation procedure. Used all of the tranformation products on the plate.

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