Having difficulties with your experiment?

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View the relevant questions below:

 

Here are some things to consider:

  1. If the OD of cells that are used is too high, they will not spheroplast. Do not overgrow cells. 
  2. Do not use old cells and make sure that they are in log phase of growth.
  3. Make sure to mix zymolyase well before using. Zymolyase is more of a suspension than a solution. 
  4. Make the PEG solution fresh each time and check the pH.
  • Make sure that you have harvested cells during log-phase growth (OD <1.0 generally).
  • If electroporation is being used, see the electroporator manual for suggested conditions. Vary electroporation parameters if necessary.
  • Use more DNA.
  • Use freshly made competent cells.
  • If the LiCl transformation method is being used, try boiling the carrier DNA.

Here are possible causes and solutions:

Cause

Solution

The pH of Solution I or Solution III may have drifted. The pH of both
solutions should be 8.0

Check the pH of Solutions I and III. If the pH is low, increase it by adding NaOH. If the pH is high, decrease it by adding HCl. Store solutions at 4°C in order to minimize drift in pH.

Transformation reaction not mixed
during incubation

Be sure to mix the transformation reaction every 15 minutes throughout the 1 hour incubation at 30°C. Vortexing works best.

Incubation time is too short or temperature is too low

Pichia pastoris transformations may be incubated for longer periods of time (up to 3 hours) and at higher temperature (35–37°C). This may, in some instances, result in higher transformation efficiencies.

Cell density is too low (OD600 <0.6)

Resuspend cells from Preparation of Competent Cells, Step 6, page 1 of the manual, in a smaller volume (i.e.,500 μL).

Generally, large colonies represent transformants containing pPIC6/pPIC6α integrants, while small colonies represent transformants containing pPIC6/pPIC6α non-integrants. These non-integrants have transduced the pPIC6/pPIC6α plasmid, and therefore, exhibit a low level of blasticidin resistance in the initial selection process. Upon subsequent screening, these non-integrant transformants do not retain blasticidin resistance.

When choosing a blasticidin-resistant transformant for your expression studies, we recommend that you pick blasticidin-resistant colonies from the initial transformation plate and streak them on a second YPD plate containing the appropriate concentration of blasticidin. Select transformants that remain blasticidin-resistant for further studies.

When using the pPink-HC vector, the transformation efficiency of the host strain may appear low because colonies with only a few copies of the marker will not produce enough ADE2 gene product to grow and will be selected against on medium lacking adenine (i.e., adenine dropout medium or minimal medium). Only colonies that have integrated multiple copies of the ADE2 marker will able to grow without adenine. Since the gene of interest is linked to the selection marker, the white colonies could also result in higher expression of the gene of interest.

This is likely due to low inoculum. We recommend having a starter culture to use, as it is more accurate than using a colony. You can use the doubling time to calculate the starting OD. In YPD, the doubling time is about 1.5 hours. In MGY, the doubling time is going to be about 3 hours.

Here are possible causes and solutions:

Problem

Cause

Solution

No multimers or low number of multimers after transformation into E. coli

CIAP defective

 

 

Not enough insert DNA to ligate

Construct is unstable in E. coli

 

Multimers are too long to ligate efficiently

Use fresh CIAP.
Add more CIAP. Add 1 unit of CIAP and incubate 15 more minutes at 37°C. This is somewhat risky as CIAP can degrade the ends of your DNA.

Add more BamH I-Bgl II expression cassette to your ligation.

Decrease the number of cassettes in the vector.

Try ligating each expression cassette
Stepwise.

Recombinant vector rearranges and deletions are detected

Construct is unstable
in E. coli

Decrease the number of cassettes in the vector.

No antibiotic-resistant Pichia transformants

Integration efficiency is low

Transform using more DNA and/or
do multiple transformations with more DNA and cells.

Here are possible causes and solutions:

Problem

Cause

Solution

No multimers or low number of multimers after transformation into E. coli

CIAP defective

 

 

Not enough insert DNA to ligate

Construct is unstable in E. coli

 

Multimers are too long to ligate efficiently

Use fresh CIAP.
Add more CIAP. Add 1 unit of CIAP and incubate 15 more minutes at 37°C. This is somewhat risky as CIAP can degrade the ends of your DNA.

Digest more pAO815 containing one copy of the expression cassette.

Use the in vivo method to isolate multimers.

Try ligating each expression cassette separately.

Recombinant vector rearranges and deletions are detected

Construct is unstable
in E. coli

Use the in vivo method to isolate multimers.

Pichia His+ transformants
do not have multimers

Vector was linearized with the wrong enzyme (restriction enzymes in the
AOX1 region are duplicated when multimers are created)

Linearize your construct with Sal I or Stu I to insert the construct into
his4.
Analyze your construct for other unique restriction sites in the vector backbone that are near the 5´ AOX1 region or the 3´ AOX1 region. These sites will preserve your multimers and allow recombination with AOX1.

Here are common troubleshooting scenarios with possible causes and solutions:

Problem

Cause

Solution

False positives

Not enough levels of Geneticin® resistance

Colonies can appear to be Geneticin® resistant, but are actually not. To prevent this, purify your putative Geneticin®-resistant colonies and confirm the level of Geneticin® resistance observed for each colony before proceeding further.

Low number of Geneticin®-resistant colonies isolated

Not enough His+ transformants were screened

The frequency of spontaneous, multiple-integration events occurs only at a rate of 1–10%. You will need to screen thousands of His+ transformants to isolate an optimal amount of colonies.

Few recombinants with gene of interest

Not enough His+ transformants were screened

You will need to screen thousands of His+ transformants to isolate an optimal amount of recombinants with the most copies of your gene, as successive multiple insertions are more rare.

Low isolation of His+ transformants

Low transformation efficiency

Using electroporation instead of spheroplasting may increase the transformation efficiency, thereby allowing you to isolate more His+ transformants.

Here are possible causes and solutions:

Cause

Solution

Transformation reaction not mixed
during incubation

Be sure to mix the transformation reaction every 15 minutes throughout the 1 hour incubation at 30°C. Vortexing works best.

Incubation time is too short or temperature is too low

Transformations may be incubated for longer periods of time (up to 3 hours) and at higher temperature (35–37°C). This may, in some instances, result in higher transformation efficiencies.

Cell density is too low (OD600 <0.6)

Resuspend cells from Preparing Competent Cells, Step 8, page 2 of the manual, in a smaller volume (i.e.,500 μl).