Protocol used for Gibson

(i) Modifications
V_insert = x
V_vector = y
V_gibson = x+y
V_water = 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.

Transformation

E.coli TOP10: One Shot® TOP10 E. coli are provided at a transformation efficiency of 1 x 109 cfu/µg supercoiled DNA and are ideal for high-efficiency cloning and plasmid propagation. They allow stable replication of high-copy number plasmids.
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

PCR

The goal of PCR is to amplify a section of DNA of interest for DNA analysis (e.g. gene insertion, sequencing, etc). The amplification rate is exponential.

• Tag polimerase (25µl reaction)
Protocol Here
• Phusion polymerase (20µl reaction?):
Protocol Here

Gel

For making a small 1% gel:

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.

Miniprep

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

INTERLAB

96-Well Transformation Protocol:
Protocol
Plate reader protocol: Protocol