A) Assemble a 40uL digestion using 10x Hind Ill digestion buffer. 1 uG of the la

Question

A) Assemble a 40uL digestion using 10x Hind Ill digestion buffer. 1 uG of the lambda phage DNA (concentration 565ng/UL, and 2U if Hind III (stock solution 0.4 U/uL in 1x Hind Ill enzyme digestion buffer). 46? 11 DN HuL 10X Hind IL.dig buffer a9a3 Wate 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 ing/1mL = ooo 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

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

                                = 1.77 ul

2 U/ 0.4U/ul = 5 ul

40 ul/10X = 4 ul

therefore for the 40 ul reaction

DNA                                      1.77 ul

10 X HindIII buffer           4 .00 ul

Enzyme                                                5 .00 ul

Milli q water                      29.23 ul

Total                                      40.00 ul

B

Glycogen 1 mg / ml = 1000

10ug/1000ugX0.01 = 10 uL glycogen

40.00 ul digested lambda DNA

10.00 ul Glycogen

100.00 ul TE buffer

50 ul NH4OAc

400 ul EtOH

C

1 ul 6Xloading buffer

5 ul of loading buffer

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