Biotechnology (W16) - Week 3,4 - Day 5 Protocol: Upward DNA capillary transfer Flashcards
What will be used as the solid support for the transfer?
Larger gel apparatus.
What is done with the wick papers?
Place 2 wick filter papers into a large tray containing 10X SSC. Allow it to absorb the buffer for 60 seconds and form a capillary wick by hanging the latter over the sides of the solid support into the transfer buffer reservoirs.
What is added to the buffer reservoirs?
10X SSC transfer buffer.
What is 10X SSC transfer buffer?
SSC (Saline Sodium Citrate) Buffer 10x liquid is a standard reagent in Southern and Northern hybridization procedures. It is used to control stringency of wash buffer for the washing steps after hybridization.
How much transfer buffer is added to the reservoirs?
up to 1 cm below the top of the solid support.
What is done after the blotting paper is thoroughly wet?
Smooth out all the air bubbles using a tube.
When placing the agarose gel, where should the cut end of the gel be?
Right hand side bottom corner. Should be centered on the wet blotting paper.
What do you need to make sure there is none of when placing the gel on the wet blotting paper?
No bubbles.
What is placed on-top of the gel?
Pre-incubated membrane.
When placing the membrane, what must be considered?
Place so corners are aligned. To avoid bubbles, place one corner of the membrane to the gel and gently lower the membrane onto the gel.
What is very important once the membrane has been applied to the surface of the gel?
Do NOT move the membrane once it has been applied to the surface of the gel!
What is placed on top of the wet membrane?
3 pieces of thick blotting paper in 10x SSC
What is placed on top of the blotting papers?
Stack of pre-cut paper towels
What is the purpose of Saran wrap?
Wrap the apparatus in it to prevent buffer evaporation.
After wrapping in saran wrap, what is done?
Placed a weight on top, centered, and place initials and section number on a piece of tape and label the side of the apparatus.
