A) Assemble a 40ul digestion using 10x Hind Il digestion buffer. 1 uG of the lam

Question

A) Assemble a 40ul digestion using 10x Hind Il digestion buffer. 1 uG of the lambda phage DNA (concentration 565ng/UL and 2U if Hind II (stock solution 0.4 U/uL in 1x Hind III enzyme digestion buffer). 46? 11 DNA lo B) After a minimal incubation time of 15 minutes take out your digest and split it into 2 halves. Set one half a side. Take the other half and perform an EtOH precipitation. If you would like to add glycogen (1mg/mL) you need a final amount of 10 uG. Bring up the volume to 150 ul using TE buffer. Add the necessary amounts of 7.5M NH40Ac and EtOH Glycogen img/imL 1o00 C) Add the appropriate amount of 6X loading buffer of your DNA digest and load them onto a 0.7% agarose gel DNA digest EtOH precipitation uL 6X loading buffer ul laoding buffer

Explanation / Answer

A.

1ug / 565 ng/ul = 0.0017 ul X 1000

= 1.77 ul

2U/0.4 U/ul =5.00 ul

40 ul/10 X= 4 ul

therefore for 40 ul reaction

DNA                                       1.77 ul

10 X Hind III buffer              4.00 ul

Enzyme                                   5.00 ul

Milli Q water                           29.3 ul

Total                                     40.00 ul

B

Glycogen 1 mg/1ml = 1000

10 ug/1000ug

= 1000ug/1000 ul

= 1ug/ul

= 10 ul glycogen

40.00 ul digested DNA

10.00 ul Glycogen

100.00 ul TE Buffer

50.00 ul NH4OAc

400.00 ul EtOH

C

1 ul 6X loading buffer

5 ul loding buffer

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