Gel product purification (Promega Wizard™ Kit)
Primer working stock preparation
PCR Using Q5® High-Fidelity DNA Polymerase
Please note that protocols with Q5 HighFidelity DNA Polymerase may differ from protocols with other polymerases. Conditions recommended below should be used for optimal performance.
25 µl reaction
50 µl reaction
5X Q5 Reaction Buffer
10 mM dNTPs
10 M Forward Primer
10 M Reverse Primer
Q5 DNA Polymerase
5X Q5 High GC Enhancer (optional)
Nuclease Free Water
Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.
Competent Cells Preparation
OD value = 600nm Abs reading x 10
*This takes approximately 16 hours.
*Controlling the temperature makes this a more reproducible process, but is not essential. Room temperature will work. You can adjust this temperature somewhat to fit your schedule. Aim for lower, not higher OD if you can't hit this mark
*Try to get the weights as close as possible, within 1 gram
-Pipet buffer against the wall of the centrifuge bottle to resuspend cells. Do not pipet directly into cell pellet!
-After pipetting, there will still be some residual cells stuck to the bottom. Swirl the bottles gently to resuspend these remaining cells
-If using multiple flat bottom centrifuge bottles, combine the cells post-resuspension
-Use a mixture of 200 μl SOC and 50 μl CCMB80 buffer as the blank
-Make labels for aliquots. Use these to label storage microcentrifuge tubes/microtiter plates
-Prepare dry ice in a separate ice bucket. Pre-chill tubes/plates on dry ice
-Flash freezing does not appear to be necessary
-Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles
-Good cells should yield around 100 - 400 colonies
-Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA
We expect that the transformation efficiency should be between 1.5x108and 6x108 cfu /µgDNA
Chemical Transformation of E. coli
Ligation Protocol with T4 Ligase
As a starting point, usually people try a 1:3 vector to insert ratio first. This ratio is about the number of fragments and as fragments of vector and insert are most likely of different lengths, it is not enough to just use the same volumes of an identical DNA concentration for both, i.e. 5 µl of a 10 ng/ µl digested vector backbone of 5000 bps will have less ‘DNA pieces’ than 5 µl of a 10 ng/ µl digested insert of 1000 bps. The overall DNA amount would be the same, but there would be 5 times more insert fragments than vector fragments in this example.
ng insert = (ng vector x bps insert) /bps vector x (insert/vector) molar ratio
Make sure that you leave the T4 Ligase in the freezer until you need it. Only then do you bring it to your place in the yellow enzyme bucket and as soon as you have pipetted the ligase to your reaction you bring the yellow bucket with the ligase back to the freezer!! Same as for Q5 polymerase!
20 μl REACTION
to 20 μl
T4 DNA Ligase Buffer (10X)
linearized vector DNA
X μl for 50 ng
linearized insert DNA
X μl for 1:3 ratio with vector
T4 DNA Ligase
The Gibson Cloning Master Mix consists of three different enzymes within a single buffer. Each enzyme has a specific and unique function for the reaction:
Plasmid Isolation (Promega PureYield™ Plasmid Miniprep Kit
Plate-reader Preparation to Measure Growth in Culture
DNA/RNA Concentration Measurement using Nanodrop™ Spectrophotometer
Separation of PHB into secreted and intracellular fractions
Purification of secreted PHB pellet
NOTE: Scale reagant volumes proportionally for higher volumes of culture from which the secreted fraction was obtained. All steps should be carried out with 1/10 volume of culture.
Sodium hypochlorite extraction of PHB from Escherichia coli
NOTE:Scale reagant volumes proportionally for higher volumes of overnight culture. All steps should be carried out with 1/10 volume of the overnight culture.
For the identification of the PHA, a slight modification of the gas chromatographic method of Huijberts et al., 1994 was made.