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Contents 1 PCR purification 1.1 Aim 1.2 Materials and Protocol 2 DNA ligation 2.1 Aim 2.2 Materials and Protocol 3 Transformation 3.1 Aim 3.2 Protocol 4 QIAquick Gel Extraction Microcentrifuge 4.1 Protocol

PCR purification

Aim

To purify the amplified fragment from PCR by using QIAquick PCR purification kit.

Materials and Protocol

Please refer to: PCR purification for materials required and the protocol.

DNA ligation

Aim

To ligate different fragments of DNA which either has similar sticky or blunt ends.

Materials and Protocol

Please refer to: DNA Ligation for materials required and the protocol.

Transformation

Aim

To insert a vector or a piece of DNA into Bacillus subtilis.

Protocol

Please refer to: Transformation of Bacillus subtilis for materials required and the protocol.

QIAquick Gel Extraction Microcentrifuge

Protocol

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
2. Weight the gel slice and add 3 volumes of buffer QG to 1 volume of gel (100 mg ~ 100 µl)
3. Incubate at 50°C and invert the tube gently at regular intrerval until the gel has completely dissolved
4. After the gel has dissovled completely, check that the color of the mixture is yellow
5. Add 1 gel volume of isopropanol to the sample and mix
6. Place a QIAquick spin column in a 2 ml collection tube
7. To bind DNA, apple the sample to the QIAquick column and centrifuge for 1 min
8. Discard the flow through and place the QIAquick column back into the same tube (Maximum volumn is 750ul)
9. Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min and discard the flow through and place the QIAquick column back into the same tube
10. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min and place the QIAquick column back into the same tube
11. Centrifuge the column for a further 1 min
12. Transfer the column into a clean 1.5 ml micriocentrifuge tube
13. To elute DNA, add 30 µl of Buffer EB to the center of the QIAquick membrane and allow to stand for 1 min
14. Centrifuge the column for 1 min and transfer the eluate to a clean 1.5 ml micriocentrifuge tube