pLenti6.2-GW/EmGFP Expression Control Vector

This can happen due to excess blasticidin used during selection. Determine the antibiotic sensitivity of your cell line by performing a kill curve. Use the minimum antibiotic concentration required to kill your untransduced cell line.

Promoter silencing or incorrect filter used/detection parameters for flow cytometer are incorrect could lead to no epxression of EmGFP after stable transduction. Screen for multiple antibiotic-resistant clones and select the one with the highest expression levels. Make sure the correct filter is used as well as the FITC detection parameters.

Poor expression could result from low transduction efficiency, too low of a MOI, or cells harvested too soon after tranduction. Ensure that cells are tranduced in the presence of Polybrene reagent. Check the MOI and/or use a higher MOI, and do not harvest cells until 48-72 hrs post-tranduction.

Here are some possible causes and solutions:

- Incorrect filter or incorrect detection parameters for flow cytommetry; make sure you are using a FITC or Omega XF100 filter set on yoru inverted fluorescence microscope or the FITC detection parameters on your flow cytometer.
- Viral stock stored incorrectly; aliquot and store at -80 degrees C, do not freeze/thaw more than 3 times.
- Polybrene reagent not included during transduction; transduce pLenti6.2-GW/EmGFP into cells in the presence of Polybrene reagent
- Too soon to see EmGFP expression; wait 4 days post-tranduction

There is no theoretical limit to insert size for a BP reaction with a pDONR vector. Maximum size tested in-house is 12 kb. TOPO vectors are more sensitive to insert size and 3-5 kb is the upper limit for decent cloning efficiency.