Protocol used for Gibson
(i) Modifications
Vinsert = x
Vvector = y
Vgibson = x+y
Vwater = 0µl
(ii) Incubation for 1h, not 15 min
(iii) Before transformation: One transformation with x ul concentrated Gibson solution and one transformation with
Gibson solution diluted 1:3 and transformation with 3*x ul diluted Gibson solution.
Chemical Transformation Procedure Modifications In Step 5, Incubate for exactly 30-45 seconds in the 42°C water bath. Do not mix or shake. In Step 7, Add 200-250 µl of rom temperatured S.O.C medium to each vial. S.O.C is a rich medium; sterile technique must be practiced to avoid contamination
E.coli DH5Alpha: Modifications Step 15 and Step 16 not done
- Tag polimerase (25µl reaction) Protocol Here
- Phusion polymerase (20µl reaction?): Protocol Here
- Weigh out 0.5 g of agarose and mix it with 50 ml of 1x TAE buffer in a 100 ml Erlenmeyer flask.
- Dissolve the agarose by bringing the mixture to the boiling point in a microwave oven, followed by mixing (by swirling the flask). Repeat the heating and mixing until all the agarose has dissolved.
- Cool the agarose solution to ~50 o C by leaving it on the bench for ~20 min (or you may accelerate the cooling by applying cold water from the tap to the outside of the flask).
- Using gloves, add 5 l GelRed (10 000x). Swirl the flask gently to mix, try to avoid bubbles.
- Pour the gel carefully into the mold. Bubbles may be removed/punctured by using a pipette tip.
Protocol Modifications:
- During the first attempt ethanol was not added to the PE buffer, which resulted in an unsuccessful miniprepl
- In the second attempt 72/4% ethanol was added, as opposed to the recommended 96-100%, resulting in a successful miniprep
Protocol
Plate reader protocol: Protocol