Gel Extraction and C1V1 calculations for Isolate Sample Dilutions

 

After last weeks ligation failure and being unable to find a new ligation procedure, its been decided to put the restriction enzyme ligation project on pause. For now I'm helping out the overlap group again, which really just means helping Lani with whatever project she's currently doing. 

While Monday was spent trying to figure out the restriction enzyme project, Wednesday I helped with a gel extraction and Friday we spent doing C1V1 calculations.


Gel Extraction Procedures- 

    We started by removing the DNA fragment from the gel, putting it in a microfuge tube and weighing it.

    After we added the gel dissolving buffer, with 400 microliters of buffer per 100 milligrams of the gel

    We then incubated the sample at 50 degrees Celsius until it was dissolved, inverting it slightly every so often

    Then we inserted the column into a collection tube and loaded the sample. After spinning for a minute we discarded the flow through

    We then reinserted the column into a collection tube and added 200 microliters of DNA wash buffer, and let it spin for a minute

    After that we once again inserted the column into a collection tube and loaded the sample. After spinning for a minute we discarded the flow through

    We transferred the column into a clean microfuge tube, and added 6 microliters of DNA elution buffer after waiting a minute and we spun it for one minute to elute the DNA.


For the C1V1 calculations, we were given the readings of isolate samples and had to calculate how to dilute them to 80 ng, at either 20 μl for the final measurement if the sample was below 840 ng, or with the initial sample measuring 2 μl if it was above 840 ng. 




Table of Calculations - 

Isolate Sample Label

Initial Concentration

V1 Calculation (μl)

V2 Calculation (μl)

P1

560 ng

2.857

-

P2

1.4 μl

-

35

P7

830 ng

1.927

-

P17

580 ng

2.758

-

P21

830 ng

1.927

-

P22

860 ng

-

21.5

P23

920 ng

-

23

P34

229 ng

6.987

-

P41

540 ng

2.960

-

P43

1.4 μl

-

35

P65

720 ng

2.22

-

P71

1 μl

-

25

P72

241 ng

6.639

-

P74

540 ng

2.96

-

P76

154 ng

10.389

-

P79

2 μl

-

50

P80

1.4 μl

-

35

P81

1.5 μl

-

37.5

FR100

2 μl

-

50

Table of Amounts for Dilutions -

Isolate Sample Label

Amount of sample (μl)

Amount of buffer (μl)

End volume (μl)

P1

2.9

17.1

20

P2

2

33

35

P7

2

18

20

P17

2.8

17.2

20

P21

2

18

20

P22

2

19.5

21.5

P23

2

21

23

P34

7

13

20

P41

3

17

20

P43

2

33

35

P65

2.2

17.8

20

P71

2

23

25

P72

6.6

13.4

20

P74

3

17

20

P76

10.4

9.8

20

P79

2

48

50

P80

2

33

35

P81

2

35.5

37.5

FR100

2

48

50




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