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Griffin:shRNA Transfection: Difference between revisions
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'''Note:''' Due to the random integration of the lentivirus into the host genome, varying levels of shRNA expression may be seen with different colonies. Testing a number of colonies will allow the optimal degree of expression to be determined. | '''Note:''' Due to the random integration of the lentivirus into the host genome, varying levels of shRNA expression may be seen with different colonies. Testing a number of colonies will allow the optimal degree of expression to be determined. | ||
=====Abbreviated Transduction Protocol===== | |||
*'''Day 1:''' | |||
Plate continuous (immortal/transformed) cells (1.5x10^5) in 6 well dishes in 3mL of normal growth media. | |||
*'''Day 2:''' | |||
Rinse cells, and add back 1mL RPMI/10%heat inactivated FCS/ penn/strep. | |||
Add 1uL of 1000x polybrene (Millipore). | |||
Add 30uL of virus (MOI=1; 5e6 IFU/ml * 30 uL = 150,000 particles). | |||
*'''Day 3''' | |||
Rinse off virus and replace with normal growth cell media. | |||
*'''Day 4''' | |||
Add selection (1ug/mL Puromycin), or split cells as needed into flasks containing selection. | |||
After two weeks, in which non-infected cells die off in selection media (control wells that were not treated with virus) | |||
Plate cells for RNA isolation and PCR for target gene. | |||
==shRNA Controls== | ==shRNA Controls== | ||
