Protocol and Mechanism

Vector amount comparison

Insert/Vector Ratio check

Assemble Reaction Time Check

Reaction set up temperature effect

Hot-start feature

Homology Length Check

Insert Size Check

DNA End type check

Vector from Restriction Enzyme Digestion

End Type comparison from restriction enzyme digestion

Insert Source check

Crude PCR product usage

Master Mix Storage Stability

3- pieces assembly

3 + insert cases

Transformation Enhancer

Transformation Enhancer Mix Time

Expression Examples

5th Group vectors

6th Groups Vectors

7th Group Vectors

Competent Cells

Competent Cells Sauce Check

Competent cell preparation method comparison

Heat Shock Time Optimization

ZY Cloning vs Direct Cloning

Antibiotic Concentration on plate check

Rapid thawing of Competent cell

Mung Bean as plating tool

Competent cell type check

If Switching from other cloning systems

Expression conditions

The recommended expression conditions for ready to use vectors.

1.   The strain for plasmid production is DH10B. DH10B is also good for the protein expression with vectors with high repetition sequences.  The strain for pBAD promoter expression is DH10BC. The strain for T7 promoter expression is DH10BT7.

2.   Expression from the intein vectors, a temperature shift to 25 oC is recommended when induced. These vectors include pKBHMD5, pKBHND5, pKBHPD5, pKBHTD5, pKBHXD5, pKBHTD6 and pKBHXD6. Also, the expression media containing glycerol is not recommended for the intein vectors for the culture tends to be acidic and cause high in vivo cleavage.

3.   Procedure steps are: Inoculate the overnight culture with colonies or cell stock, grow overnight culture which generally prefer at 25 oC, then the large culture is inoculate with 2% volume of overnight culture, grow to late log phase such as 5 hours at 37 oC, induce with the appropriate inducer, with temperature shift if needed.

4.   While general binding at 100 mM, pH8.5, Kpi and 10 mM Imidazole buffer, the binding affinity of each tag to the IDA nickel magnetic beads, are as following measured in imidazole-Acetate, pH 7.0:

a.       His tag only, including pKBH5, pKBH7, pKT7H7, and the ones with short additional amino acids such as pKBaH7 and pKBtH7, varies with the actual expressed protein.

b.       Weak binding group: His-MBP, His-NusA, elution starts at 31 mM Imidazole-Acetate, pH 7.0.

c.       Medium binding group: His-SUMO, His-P17, His-Trx, elution starts at 125 mM Imidazole-Acetate, pH 7.0.

d.       Strong binding group: His-GST, His-NEXT, elution starts at 250 mM imidazole-Acetate, pH7.0.

e.       Composite tags count the first one for binding expectation.

5.   Intein tags generally don’t need to be eluted with variation of imidazole concentration, the cleavage buffer can be 100 mM NaPi, pH6.0 or Tris-HCl, pH 6.0 with 500 mM NaCl. The cleavage efficiency at 4 oC is 70% to that of at 25 oC.

Site-Directed Mutagenesis 

Trouble Shooting

Trouble Shooting:

1.   If there are no colonies after the transformation of the assembly reaction:

a.       Ensure both the 3x ZY Cloning Master Mix and the Competent Cell transformation control were successful.

b.       Evaluate the homology region. In some cases the homology region is too short. Additionally, avoid sequences which may be self-complimentary.

c.       Double check the antibiotic concentration as 40% of the normal concentration.

d.       Ensure sufficient competent cell volume is added. At least 50 μl is required for the 3 μl reaction mix.

e.       Use gel electrophoresis to check the DNA quality of the vector to make sure it is not degraded.

2.   If there are too many colonies present in the negative control:

a.       Leftover uncut vector from restriction enzyme digestion

b.       Template DNA in PCR is too high concentration or the DpnI digestion to remove the template after PCR was insufficient.

c.       Contamination may have occurred, particularly from supercoiled DNA with the same antibiotic resistance.

d.       The antibiotic in the is too old or the plate was prepared with too low concentration of the correct antibiotic.

3.   If there are too many colonies without correct insert:

a.       Make sure the insert is pure and high quality, whether generated from PCR or gene synthesis. Use an agarose gel to visualize insert quality.

b.       For PCR-generated DNA, column purification may not remove undesired small size fragments. If so, use gel purification to purify further.