4, 20 and 25 |
Incomplete restriction digestion: 3 fragments are seen instead of 2 |
Too much DNA used for the restriction digestion |
Dilute the plasmid DNA and reset the digestion reaction or incubate longer times until the digestion is complete |
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|
The purified plasmid DNA contains residual cellular proteins and needs to be cleaned up |
Perform additional standard phenol/chloroform extractions followed by ethanol precipitation using salts52 before resetting the digestion |
|
|
Enzyme(s) are old and less efficient or inactive |
Add more enzymes and incubate for longer duration. |
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|
Purchase new enzymes if inactive |
10, 24 and 31 |
Few or no colonies |
Cells used for transformation have lost competence |
Change the method of preparation of competent cells and use the original plasmid vector to check the efficiency of the competent cells |
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|
Low transformation efficiency |
The time constant must be 4–5 for maximum efficiency |
14 |
The recombinant plasmid contains no insert |
A contaminant colony was selected |
Discard the negative clone and screen more colonies. If the problem persists, step back to the previous valid clone and restart the experiment from there |
|
The recombinant plasmid contains an insert with errors in its sequence |
Possible mutations occurred during cloning |
Discard the clone and restart the cloning from the previous step |
|
The recombinant plasmid contains an insert of the wrong size |
Possible recombination/deletion occurred during cloning due to the repetitive nature of the insert |
Discard the clone and restart the cloning from the previous step |
36 |
Low expression efficiency |
mRNA secondary structure formation |
Optimize spider silk codon usage for E. coli in design avoiding G- and C-rich codons |
|
|
Bacterial clone with low expression level |
Screen colonies in the presence of IPTG in culture medium. The smallest colonies usually give highest expression level |
52 |
Total protein not binding to the resin |
Either the resin is old or the binding buffer may not have been added to the protein extract. The buffer composition and/or pH are wrong |
The binding buffer should be at least in 1:1 ratio with the protein extract. Check the buffer composition. Replace the resin if the problem persists |
|
Protein of interest not binding to the resin |
Possible change in the conformation of the protein concealing the His-tag or loading too much of the total silk protein |
Analyze the flow-through and wash fractions to see if the protein of interest is lost during binding or washing. |
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|
|
To avoid conformation problems, engineer a C-terminal His-tag |
|
No protein elution peak |
The concentration of imidazole in the elution buffer is too low |
Increase the concentration of imidazole and use gradient elution |
|
Too many proteins are present in the eluted fraction |
Nonspecific elution of proteins |
Increase the washing time and/or decrease the elution buffer concentration |
|
Degradation of products after purification |
Possible proteolysis |
Maintain low temperature during the entire extraction and purification processes. Add protease inhibitors to the purification buffers |
71 |
No signal on the X-ray film |
Poor transfer or no transfer |
Stain the gel with Coomassie stain following transfer to determine if transfer is complete. |
|
|
|
Increase or decrease the transfer time. The duration may vary depending on the protein size. Higher molecular weight proteins require longer transfers and low molecular weight proteins require shorter transfer |
|
|
No expression |
If the protein marker gives a positive signal but the recombinant proteins are not detectable, verify the identity of the recombinant plasmid clone. The frozen stock may have been contaminated |
|
|
Antibody or substrate not working |
Some of the proteins in the marker are also His-tagged and serve as a positive control. Replace the antibody or substrate if there is no signal from the protein marker |
|
|
Insufficient chemiluminescent substrate or incubation time too short |
Add more substrate and increase the time of incubation |
|
High background and low signal |
Inadequate blocking or insufficient washing |
Change the blocking agent to bovine serum albumin. Block with 5% (wt/vol) milk and wash stringently by increasing the amount of Tween-20 in the 1× PBST |
73–75 |
Clog of PEEK tubing |
Presence of insoluble protein in spinning dope |
Dissolve the protein in HFIP and vortex for a longer time. You can also crush the protein agglomerate with a pipette tip |
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|
Presence of residual salts in the lyophilized protein due to inefficient dialysis |
Increase dialysis time and number of buffer changes |
|
No silk formation |
Low protein concentration in the spinning dope |
Make sure to have a 25–30% protein solution for the spinning dope |
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|
Inadequate solvent for spinning dope preparation or for the coagulation bath |
Increase or decrease the amount of water added to the spinning dope. If fibers still do not form, try different alcohols or organic solvent (acetone) as coagulants |