Refrain from repeated freezing and defrosting as antibodies lose activity and start to agglutinate leading to blockages of the injection capillaries.
Defrosting should be performed as quickly and gently as possible.
Before loading the capillary, centrifuge material for 15 minutes at 4°C (10,000 x g). Chill supernatant or load directly into capillary.
DNA [5]
Purification The highest expression level of microinjected plasmid DNA is achieved when the DNA has been purified by CsCl ethidium bromide gradient centrifugation in accordance with the Maniatis protocol [6].
Dissolve DNA in bidist. water after purification.
Buffers, e.g., PB’s, are not recommended as this often leads to blockages of the capillaries.
DNA concentrations of 20 µg/ml to 200 µg/ml for plasmid DNA injection has been recommended [1].
Storage Store at –20°C in small aliquots of 5–10 µl.
Defrosting should be performed as quickly and gently as possible.
Before loading the capillary, centrifuge material for 15 minutes at 4°C (10,000 x g). Chill supernatant or load directly into capillary.
Repeated centrifugation does not cause damage but is not necessary for each capillary filling when chilled with ice and used within an hour.
RNA Purification
Any standard protocol is suitable for purifying RNA solutions [6].
As for DNA, RNA is dissolved in bidist. water after purification.
Concentrations of 1–2 µg/ml should be used for mRNA and up to 10 µg/ml for total RNA.
Storage Store at –80°C in small aliquots of 5–10 µl.
For longer periods, it is advisable to dissolve and store the cleaned RNA in alcohol instead of water.
Defrosting should be performed as quickly and gently as possible.
Before loading the capillary, centrifuge material for min. 15 minutes at 4°C (10,000 x g). Chill supernatant or load directly into capillary.
Only centrifuge and use each aliquot once.
Dyes, fluorescent injection markers [1]
Successful fluorescent injection markers used to identify and follow injected cells are: fluorescence labeled dextrans, antibodies, bovine serum albumin.
Dextran marked e.g. with rhodamine of a concentration of 2 µg/ml can be detected for up to 48 hours in the cell.
Fluorescein fades with time and results in harmful radicals, therefore markers should preferably be labeled with rhodamine.
Storage Store at –80°C in small aliquots of 5–10 µl.
Many solutions are sensitive to light. Thus, exposure to direct light should be avoided.
To avoid blocking of the capillary during microinjection, solutions containing the markers should be filtered with a syringe filter (pore size 0.2 µm) whenever possible. Also, before loading the capillary, the sample should be centrifuged for 15 minutes at 4°C (10,000 x g).
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