Transgenic Core

Purification of DNA for Microinjection

QIAquick Gel Extraction Kit Protocol for DNA Purification

1- Digest 20ug of plasmid DNA as necessary to cut apart the fragment and the DNA.

2- Run on a 1% agarose gel until bands are well separated. Check quickly with a long wave UV light, keeping exposure to a minimum so as not to damage the DNA.

3- Use a sterile scapel to excise the desired band from the gel. Place get slice in a 1.5 ml eppendorf tube.

4- Weight the gel slice. Place in a larger tube if necessary so that 3 volumes of Buffer QG can be added per 1 volume of gel (100mg=100ul). Add 300ul per 100mg gel.

5- Gently mix until gel slice has dissolved completely. The buffer should still be yellow. If the fragment size is <500bp or >4000bp add 1 vloume of isopropyl alcohol and mix gently.

6- Divide the mixture equally into two QIAquick columns (700ul is maximum load per spin). Spin for 1 minute at 13,000 rpm. Discard flow-through and continue until all of the buffer mix has been applied to the columns.

7- Wash 2X with 500ul of buffer QG, discarding flow through after each spin.

8- Add 750ul of Buffer PE to the columns and let sit for 5 minutes. Spin and discard flow through.

9- Wash twice more with 750ul of Buffer PE, this time spinning immediately after adding to the columns.

10- Discard all flow through and spin for 5 minutes at 13,000 rpm to remove any remaining wash buffer.

11- Place one of the colums in a fresh 1.5ml eppendorf tube. Add 40ul of filtered TE buffer (ph 8.0) directly over the column.

12- Let stand for 1 minute and then spin for 2 minutes at 13,000 rpm.

13- Collect the flow through and add it to the second column. Let stand 1 minute and spin as before.

14- The purified DNA can then be quantified by a spectrophotometer and gel analysis.