This experiment describes the purification of Streptomyces paramagneticus anti-biotin protein beads. This experiment was derived from PCR Laboratory Guide (2nd edition) by Seed Kang and Qu Lijia.

Operation method

Purification experiments with paramagnetic streptavidin anti-biotin protein beads

Materials and Instruments

ATEN buffer Polyethylene glycol T5E5 buffer Dpn I Protease K RNAase A primer with biotin-TEG at the 5' end and ribocytosine at the 3' end Protease K-treated PCR product
Magnet Streptomyces anti-biotin protein beads

Move

I. Materials

1. Buffers, solutions and reagents

1X ATEN buffer (see step 1)

10X ATEN buffer (see recipe for Program 1)

Polyethylene glycol (30% PEG, 1.5mol/L NaCl)

T5E5 buffer (see recipe for Option 1)

2. Enzyme and enzyme buffer

Dpn I

Protease K

RNAase A

3. Nucleic acids and oligonucleotides

Primer with biotin-TEG at the 5' end and ribocytosine at the 3' end

Proteinase K-treated PCR product

4. Specialized equipment

Magnets

Streptomyces anti-biotin protein beads (Genoviskm's product is highly recommended)

II. Methods

1. The target fragment is amplified with primers with biotin-TEG at the 5' end and ribocytosine at the 3' end. after PCR, Dpn I, PEG precipitation, RNAase and Proteinase K treatments in T5E5 are performed as described in Scheme 1. The samples were finalized with 1XATEN buffer by adding 1/9 volume of 10XATEN buffer.

2. Pre-precipitate beads. Remove some Streptavidin anti-biotin protein beads in a storage tube, magnetize them, and remove the storage buffer. Wash the beads twice with 10 times the volume of 1X ATEN buffer (one of which should be soaked at 80C for 5~lOmin). Resuspend the beads in the original volume of 1X ATEN Buffer and leave at 4°C for up to 2 weeks.
One of the purposes of drenching is to remove loose streptavidin-antibiotin proteins. Residual proteinase K will not damage the streptavidin beads.

3. For each 130ul of Proteinase K-treated PCR product, add 15ul of 10XATEN buffer and incubate for 30 min at 52°C. Sometimes the previous RNAase step or the 65°C Proteinase K step will melt some of the primers. The high salt bath ensures that the primers are temporarily returned to their original sticky ends so that the biotin at the 5' end can hook them up to the beads. Without this re-annealing process, half of the products sometimes fail to stick together. Add 60 to 100 ul of the streptavidin anti-biotin protein beads prepared in Step 2 resuspended in 1X ATEN buffer, warm bath at 25°C for 30 min, magnetize them. Keep the supernatant of the beads and take some electrophoresis to test the efficiency of bead uptake.

4. Rapidly rinse the beads 1 or 2 times with 200-500ul of 1XATEN buffer to remove more of the initial PCR template and RNAase.

5. Heat elute the beads with 100ul of 1XATEN buffer 2-3 times at 80°C for 3-5 min each time. transfer the tubes to the wells of a thermal module containing a magnet at 80°C for a few minutes.

6. One at a time, one holds the warm test tube in one's hand near a magnet, or on a magnet holder, and aspirates the supernatant while it is still hot. This is the DNA of the ribose clone containing the 3' sticky end. magnetize this DNA-containing supernatant, or centrifuge it one last time to remove traces of beads, and transfer it to a new tube. Keep frozen for up to 2 weeks.

7. This DNA can be used for cloning. the DNA is now dissolved in 1X ATEN buffer to ensure that the final level of ATEN is 1X during the annealing process. begin cloning as described in Step 5 of Scheme 1.

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