IGEM:Brown/2007/Lab Protocols/Preparing Ligation Backbone and Insert

Preparing Ligation Backbone and Insert:

1. Gel isolate both backbone and insert on a preparative agarose gel with 1 ul of 10 mg/ml ethidium bromide per 25 ml of gel. Run the gel no faster than 100-125 mA.

2. Examine the gel and excise the correct bands on the preparative setting of the UV transilluminator. Put the gel slice in an eppindorf tube.

3. Weigh the gel slice. Make sure that there is no more than .3 grams of gel slice per eppindorf tube. Assume that the weight of the slice is equal to the volume (.1 gm = .1 ml).

4. Add 3 volumes of 6 M Sodium Iodide solution and heat the gel slice at 55-65 C until completely melted (Approximately 5 minutes).

5. Place the gel slice-NaI solution on ice to cool. Add well resuspended glass milk. As a general rule add 5 ul of glass milk for 1-5 ug of DNA, add an additional 5 ul of glass milk for each 1 ug of DNA in excess of 5 ug. Incubate on ice for 5 minutes.

6. Pellet the glass milk with a brief spin in the microfuge and discard the supernatant. Resuspend the glass milk in 700 ml ice cold New Wash with a blue tip. Repeat this wash two more times.

7. After the third wash discard the supernatant and respin the tube and remove as much of the new wash as possible.

8. Elute the DNA in 1X TE pH=7.5. For backbone use 50 ul of TE, for insert use 5-25 ul of TE based on the size of the insert and the estimated ug recovered. After resuspending the glass milk in TE, incubate the mixture at 65 C for 5 minutes, then spin briefly in a microfuge to pellet the glass milk. Transfer the eluate to a fresh eppindorf tube.

9. Check 2 ul of each backbone and insert on an agarose gel. Estimate the approximate concentration of the DNA in each sample.