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6.19 PEG Purification
I) Purpose
- The first step in the phage purification process. Purify phage from bacterial debris so that we can determine the banding pattern of T7 in CsCl.
II) Expected Outcome
- Phage will be purified from the bacterial debris to be further purified in a CsCl gradient.
III) Reagants Used
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- phage suspension buffer
- 10 mM Tris-HCl
- 100 mM NaCl
- 10 mM MgCl2
- DNase I
- RNase A
- chloroform
- NaCl powder
- PEG 8000
- T7 bacterial lysate
IV) Actual Procedure
- Add 166.5 µL DNase and 22.5 µL RNase to T7 bacterial lysate.
- Add .0904 mL chloroform to complete lysis and let the preparations sit for 30 minutes at room temperature.
- Dissolve 1.32 g of solid NaCl and let cool at 4◦ C for 1 hour.
- Centrifuge the suspensions at 6500 rpms (7000 g) for 10 minutes at 4◦ C. Remove supernatant and put in clean flasks.
- Dissolve 3.96 g PEG 8000 and let sit at 4◦ C overnight.
- Centrifuge the suspensions at 8000 rpms (10000 g) for 15 minutes at 4◦ C. Discard the supernatant.
- Turn the centrifuge bottles over to let sit for 5 minutes to drain off any remaining supernatant.
- Suspend the pellets in .675 mL phage suspension buffer and let sit overnight at 4◦ C.
V) Results
- We were able to purify phage to a point that it can now be used in the CsCl gradient. We finished with phage dissolved in phage suspension buffer. We will be using this solution in a later class to further purify in a CsCl gradient.
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