Calcium phosphate transfection protocol
After a suitable length of time (overnight for Calcium tolerant cells, 4-6 hours for Calcium intolerant cells remove the CaPO4 containing medium.
Since the solutions are of low ionic strength, often 2 or 3 pH meters must be consulted to sharad upadhye books rashi chakra correctly determine the.
For example, for virus-vector preps where the medium is windows defender for windows 8 turn on harvested you might want to plate more cells to increase titer.
Into one set pipet:.5ml 2X hebs.And van der Eb,.They will be 70-75 confluent the next day.).Incubate the cells at 37 C with 5 CO2 for 24 h and then replenish medium.267.96).0g Dextrose.0g hepes, weigh out ingredients, bring to 450ml with sterile distilled H2O, adjust pH to:.2 for plasmid transfections.A DNAcalcium phosphate co-precipitate forms, which adheres to the cell surface and is taken up by the cell, presumably by endocytosis.For those experiments where more transfection mix is needed, simply use a multiple of the reagents described below: For cells on 24 well plates, combine equal amounts of the plasmid in question and normalization signal with L7RH-beta-Gal plasmid.Gently agitate the dish to distribute the precipitates evenly over the cells on the plate.Total of 200 l when added new subway surfers game to above.This protocol has been optimized for transfection of neonatal rat cardiac myocytes.Mix gently but thoroughly to allow formation of precipitates evenly.Calcium Phosphate Transfection of Eukaryotic Cells.Bring to 500ml with sterile distilled H2O.Note: This is the most important step for forming DNA/calcium phosphate co-precipitate.Day Two: Transfection, to prepare the cells for transfection, add fresh complete medium according to the chart below: Culture Dish, fresh complete medium added (mL).5 cm dish 1 6 cm dish 2 10 cm dish 4, two hours later, prepare two tubes with transfection.Jiing-Dwan Lee see Chen.
Notes: After adding the 2X HBS, cap the eppendorf and let stand at room temp for no less than 15 min.
Add 40 l per well immediately after plating (20 l each of the luciferase plasmid with 20 l of the beta gal mix).
Most investigators find that it saves a lot of time and DNA if this reaction is initially performed without DNA to check that the pH of the solutions will allow the formation of a precipitate.Carry HEK-293 cells in Eagle's Minimum Essential Medium with 10 FBS.HEK-293 cells (atcc, catalog number: CRL-1573 eagle's minimum essential medium (atcc, catalog number: 30-2003 fetal bovine serum (FBS) (atcc, catalog number: 30-2020 calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670 hepes (Sigma-Aldrich, catalog number: H4034 sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 sodium phosphate dibasic (Na2HPO4).Culture cells in standard serum-containing or serum-free medium appropriate for the cell type.This protocol is also widely used for co-expression of plasmids for packaging viruses.Remove glycerol solution and wash twice with PBS: Culture Dish Volume PBS for each wash (mL).5 cm dish 2 6 cm dish 5 10 cm dish 10 Day Three: Change Medium Following overnight incubation (or optional glycerol shock aspirate medium (or PBS) and replace.Bring to total of 300 l with sterile H20.References Chen,., Lu,., Yang,., Fearns,., Yates,.While bubbling the HeBS (tube B add contents of tube A (from step 2 dropwise.
The pH should be checked with nonbleeding sterile pH sticks or reliable pH meters.