Show work 1.You need to do a transfection of your tissue culture cells, but befo

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1.You need to do a transfection of your tissue culture cells, but before you do,

you must so some prep work. Today, you have to plate your cells equally in a

6-well plate. You have to first count your cells. You trypsinize your cells and

add media to a total volume of 5mLs. You remove 20µL of those cells to a

1.5mL tube with another 20µL trypan blue.

a. You count the following live cells in the four quadrants:

i. Quadrant 1 – 168

ii. Quadrant 2 – 132

iii. Quadrant 3 – 197

iv. Quadrant 4 – 117

How many cells/mL do you have?

How many mL will you need to plate 500,000 cells into one well? Add media to 2mL of total volume.

2.Once you plate your cells, the next step is to prepare the DNA for the transfection. You read your DNA on the spectrophotometer and it tells you that you have 12µg/mL of DNA, with an A260 of .413 and an A280 of .22

a.What is your purity

b.Is this a good purity for downstream use in transfection or PCR? Provide the recommended purities for RNA and DNA.

c.What is one reason a sample could have a bad purity after isolation/purification?

3.Walk me step by step how to pour 1% gels from scratch. 2 gels = 100mL

a.Make 1L of 1x TAE from a 50x stock

b.Weigh out the agarose for 2 gels

c.How much TAE to add?

d.Heat up obviously

e.Cool down

f.Add Ethidium Bromide, how much?

i.Also note a safety precaution

g.How much DNA do I load and how much dye to add?

h.What happens if there is no dye?

i.What voltage and time do you set?

3.Make a buffer from scratch

a.1L of 5M NaCl and 1L of 1M Tris (look up MW)

b.Once your stock solutions are made make 1L of 200mM NaCl and 20mM Tris (use water to fill to 1L)

Explanation / Answer

1.

a) Total volume of cells = 5ml

Dilution factor =2:4=1:2

Total cell count in all the quadrant= 168+132+197+117

=614

Number of cells in 1ml = (Total number of cells in 4 quadrant/ 4) x 104 X dilution factor

=(614/4)x 104 x 2

=153.5 x104 x 2

=307x104 cells per 1ml

So toatl number of cells in 5ml = 5x 307x 104

  =1535x 104

  =1.535 x 107 cells

b) To re-seed 500000 cells in each well

Number of cells in 1 ml = 30.7x105

For 500000 cells= (1/30.7x105)x 500000

=5/30.7

=0.163

So to re-seed 500000 cell 0.163ml or 163ul of cells has to be taken.

2.  

Given, 260=0.413

280=0.22

purity of DNA/ RNA is defined by ratio of 260/280

=.413/.22

=1.877

a) The purity of given DNA is 1.87 which is almost pure.

b) The recommended purity of DNA =1.8(approx) and for RNA = 2 (approx). So this DNA is good for transfection.

c) Sample could be bad purity after purification due to RNA contamination.

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