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In the following reference, 1% casamino acids were used: Clare JJ et al. (1991) Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene 105(2):205-212.

In this paper, the researchers found that although Pichia grew to a similar cell density in both YP and YNB, only a very low level of mouse epidermal growth factor (0.07 µg/mL) was present in supernatants from single-copy transformants when grown in YNB, and this decreased during further incubation. By using YNB medium that had been buffered to pH 6.0 and supplemented with 1% casamino acids, secreted mEGF levels substantially increased to ~1.9 µg/mL for single-copy transformants.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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Use the following high cell density protocol for pGAP clones. Feed carbon until the desired density is reached (300 to 400 g/L wet cell weight (WCW)). If the protein is well-behaved in the fermenter, increase to 300-400 g/L WCW as with methanol inducible clones. These densities can be reached in less than 48 hours of fermentation. We have fermented constitutive expressers on glycerol using these protocols with good results. Some modifications to the Fermentation Basal Salts Medium that you might want to make are:

1) Substitute 2% dextrose for the 4% glycerol in the batch medium.
2) Substitute 40% dextrose for the 50% glycerol in the fed-batch medium.
3) Feed the 40% dextrose at 12 mL/L/hr (Jim Cregg has published data on expression using several carbon sources as substrates; dextrose gave the highest levels of expression).
4) Yeast extract and peptone may be added to the medium for protein stability.

One warning: If you are working with His- strains, they remain His- after transformation with pGAPZ. Fermentation in minimal medium will require addition of histidine to the fermenter.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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When culturing cells without complete feeds for fed-batch production, we recommend that you do not to allow glucose levels to fall below 2 g/L in culture by supplementing with glucose as needed (see product insert for more details). We have found that EfficientFeed A+, B+, and/or C+ supplements work well with Dynamis medium to maximize titers. We recommend trying EfficientFeed C+ 1X and 2X first, and if specific productivity drops off late in culture try the addition of FunctionMAX Titer Enhancer. No supplements should be added before reaching the day when half-peak cell density is reached for cultures not fed with complete feeding supplements like the EfficientFeed supplements.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

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There are several steps you can take to help ensure that the viral titer is optimized. The most common reason for low lentiviral titers is poor transfection efficiency. Only in rare instances do our own research scientists obtain a titer of less than 10e5, and in those cases, it has been narrowed down to the transfection efficiency, more specifically, because of issues with the health of the 293FT cells.

(1) Do not vortex the Lipofectamine 2000 reagent. Mix gently by inversion, and make sure it's stored properly.

(2) Plasmid DNA must be sufficiently clean for these transfections.

(3) Adhere to the recommended DNA:lipid ratio of 3:1 (9 µg packaging mix + 3 µg vector, then 36 µL of Lipofectamine 2000 reagent). If the above recommended amounts do not yield acceptable titers, you may also try the following: for each 100 mm plate, use 4.5 µg of the pLenti vector + 18 µg of the packaging mix + 67.5 µL of Lipofectamine 2000 reagent.

(4) Do not pipet up and down the lipid:DNA complexes; mix very gently.

(5) Use low-passage cells. Do not use 293FT cells over passage 20.

(6) Plate cells at 5 x 10e6 per 100 mm dish; density is very important.

(7) Make sure the cells are growing well before re-plating prior to the day of transfection. Avoid overgrowth of 293FT cells when passaging.

(8) G418 has been in the media to this point. When plating for transfection the next day, omit G418 from the media.

(9) On the morning of transfection, replace media on cells with fresh Opti-MEM + 10% FBS (about 1 hour prior to transfection, no G418). Instead of using fresh Opti-MEM + 10% FBS, you can also use complete media without antibiotics. Because the cells seem to be a little healthier, it is therefore favored by some of our researchers in R&D over the fresh Opt-MEM + 10% FBS.

(10) 24 hours post transfection, change the media. Aspirate off the media, add fresh media gently to the side of the dish, not directly to cells (serum-containing media can contain pen/strep but NOT G418).

(11) After another 24 hours (48 hours posttransfection), you can harvest the medium containing your virus. Transfected cells may be detached from the plate, or if attached should look very "plump". If most are on the plate and the cells still look healthy at this point, you can wait an additional 24 hours (72 hours post transfection) before harvesting the media (virus).

(12) To spin down the cells and harvest virus, you can centrifuge virus-containing medium at 1,500 rpm at room temperature or 4 degrees C for 5 min.

(13) Titer the virus on HT1080 cells if available. The titer can be 10-fold less if using HeLa or NIH3T3 cells.

(14) Add "non-essential" amino acids to media when working with cells at high densities.

These suggestions have helped most (if not all) customers to produce good viral titers in their own labs. We are very confident that the materials in our kits are fully functional. The components of the kits are thoroughly tested with the control lacZ vector, where we produce lacZ-expressing virus and titer it on HT1080 cells. The cut-off for the manufactured lot to pass our quality control is a titer of at least 1 x 10e5 cfu/mL. Typically the lots pass with higher titers, and we would never ship a kit that did not meet this virus production criterion. There are many things that must happen for your cells to produce high titers of virus. Cell density, health, cell cycle, lipid:DNA complexes, incubator temps, CO2 levels, etc. all contribute to good virus production. Some variables are easy to control, and some not. If you do observe low titers, it could be due to less-than-optimal conditions for some of these variables on that day for that particular experiment; and in a subsequent experiment, it is possible to achieve a higher titer.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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Below are possible reasons why you may see reduced viability following transfection, along with suggested solution.

1. Possible Cause: DNA: transfection reagent ratio sub-optimal for cell line
   Suggested Solution: Prepare complexes using a DNA (µg) to Lipofectamine 2000 (µl) ratio of 1:2to 1:3 for most cell lines. Optimization may be necessary. If so, vary DNA (µg): Lipofectamine 2000 (µl) ratios from 1:0.5 to 1:5.
2. Possible Cause: Plasmid DNA preparation contains high levels of endotoxin
   Suggested Solution: Ensure that the plasmid DNA or siRNA used for transfection is of high quality. For plasmid DNA purification kits, we recommend using our PureLink HiPureNucleic Acid Purification Kits.
3. Possible Cause: Cell density was not optimal
   Suggested Solution: Lipofectamine 2000 works best in cultures that are >90% at the time of transfection.
4. Possible Cause: Complexes were added to cells in serum-free medium
   Suggested Solution: Try using growth medium containing serum when performing transfections. Transfection performance is typically better when the cells are more viable. If you require serum-free conditions, test medium for compatibility with Lipofectamine 2000 since some serum free formulations (e.g. CD293, SFM II, VP-SFM) may inhibit cationic lipid-mediated transfection.
5. Possible Cause: Complexes not thoroughly mixed in growth medium
   Suggested Solution: Following addition of transfection complexes into medium, ensure that the plate or wells are thoroughly mixed to prevent concentration of DNA:transfection reagent complexes in the wells
6. Possible Cause: Cells have changed over time, or splitting conditions have changed
   Suggested Solution: If transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
7. Possible Cause: Antibacterial agents were used in growth medium during transfection
   Suggested Solution: Do not use antibiotics such as chloroquine, penicillin, or streptomycin in growth medium because during transfection, cells are more permeable to antibiotics, which may cause toxicity.
8. Possible Cause: Transfection reagent stored improperly
   Suggested Solution: We recommend storing transfection reagents at 4°C. Freezing of transfection reagents, or storing them at room temperature, may decrease activity.
9. Possible Cause: Cationic lipid reagent was oxidized
   Suggested Solution: Do not vortex or agitate cationic lipid reagents excessively; this may form cationic lipid reagent peroxides.
10. Possible Cause: Selection antibiotic added too soon
    Suggested Solution: When creating stable cell lines, allow at least 72 hr for cells to express the resistance gene before adding selective antibiotic.

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