Difference between revisions of "Team:UCLA/Notebook/Protein Cages/10 August 2015"
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Conclusions: Tomorrow, the SDS-PAGE will be ran in order to assess the purification. | Conclusions: Tomorrow, the SDS-PAGE will be ran in order to assess the purification. | ||
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| + | Intro: Digested and ligated Mutant #10 | ||
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| + | 10-fold Digestion: | ||
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| + | 5 uL buffer 2.1 | ||
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| + | 1 uL Xba | ||
| + | |||
| + | 1 uL Spe | ||
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| + | 1 ug DNA | ||
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| + | ddH2O (to 50 uL) | ||
| + | |||
| + | |||
| + | 37C 1 hour | ||
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| + | 80C 15 min (heat inactivation) | ||
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| + | |||
| + | Ligation: | ||
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| + | 2 uL 10x T4 ligase buffer | ||
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| + | 3:1 molar insert to vector ratio | ||
| + | |||
| + | 1 uL T4 ligase | ||
| + | |||
| + | ddH2O (to 20 uL) | ||
| + | |||
| + | |||
| + | 16C overnight | ||
| + | 65C 15 min (heat inactivation) | ||
| + | |||
| + | |||
| + | Tyler Lee | ||
Revision as of 21:06, 19 August 2015
Nithin's Notes
Ran a PCR Amplification and PCR cleanup of our PCquad 3.0 Prime. Conditions were all the same as previous PCR reactions run by Phillip.
Phillip's notes:
Introduction: Today affinity purification will be done on the cell lysates.
Procedures:
Below is a complete protocol for lysis and purification, though only purification was done today.
Cell lysis and Ni-NTA Affinity Purification
Introduction: The following procedure is for lysing bacterial cells with triton lysis buffer. For optimal activity, it is suggested that reagents be made right before lysis. This protocol is also designed for Ni-NTA affinity purification, as the lysis buffer resembles the equilibration buffer.
Triton lysis buffer: 10mL was made. Only 1mL was used, so the rest was stored at -20C.
50mM phosphate buffer (pH 8)
300mM NaCl
10mM imidazole (pH 7.4) (Optional for downstream Ni-NTA affinity purification)
1% triton x-100
Note: The above can be made and stored either in 4C or at room temperature for about a month (store in the dark if containing imidazole).
Right before cell lysis, add the following:
100ug/mL of lysozyme
One Roche EDTA-free protease inhibitor tablet per 10mL
1uL/mL of 2500U/mL DNase (adjust the solution appears viscous during the lysis).
Note: It is optimal to use the lysis buffer fresh, however this complete lysis buffer may be usable up to a month if stored in -20C with minimal thawing cycles.
-Lysis protocol:
1. Obtain the frozen cell pellet, keep on ice. 0.09g of cell pellet was lysed in 1mL of lysis buffer.
2. To the frozen cell pellet, add lysis buffer to a ratio of 1:5 grams of cell pellet to mL of lysis buffer.
3. Incubate 5 minutes on ice.
4. Vortex vigorously for 20 seconds, incubate on ice for 5 minutes. A pipette can be used to carefully re-suspend the pellet if vortexing is insufficient.
5. Repeat Step 4 two to four times, until the pellet is sufficiently broken up.
6. Centrifuge at 4C at max speed for 10 minutes. Retrieve supernatant (lysate). The pellet can be kept for downstream analysis if desired.
7. Store at -80C or -20C (-80 recommended) until use.
Ni-NTA Affinity purification (modified from the Thermo protocol):
Buffers:
1. Equilibration buffer:
a. 50mM phosphate buffer (pH 8) b. 300mM NaCl c. 10mM imidazole (pH 7.4)
2. Wash buffer: a. 50mM phosphate buffer b. 300mM NaCl c. 25mM imidazole
3. Elution buffer: a. 50mM phosphate buffer b. 300mM NaCl c. 250mM imidazole
-Procedure
Because a rotator was not available. Mixing was done by inverting in a 50mL falcon tube with ice.
1. Add one volume of resuspended Ni-resin to a container with 3-4 times the volume of resin added. 300uL of lysate was added.
2. Add two resin bed volumes of equilibration buffer, mix by rotating for 5 minutes. Ensure that the resin is fully resuspended at each step.
3. Centrifuge for 2 minutes at 700g. Discard supernatant.
4. If the above lysis buffer was not used, add protein sample to equilibration buffer at a 1:2 v/v ratio for a total volume greater than the resin bed volume if (Usually two volumes). For example, for 1mL of resin, add (1mL of protein sample + 1mL of equilibration buffer). Save at least 20uL of whole cell lysate for SDS-PAGE analysis.
5. Add protein to resin, rotate for 30 minutes. Keep cold if possible.
6. Centrifuge at 700g for 2 minutes. Remove supernatant, save for analysis.
7. Add two resin-bed volumes of wash buffer. Rotate for 5 minutes. Centrifuge. Remove supernatant. Save for analysis.
8. Repeat wash 3 to 5 times (more if necessary). 3 Washes were done.
9. Elute in one resin-bed volume of Elution buffer. Rotate for 10 minutes. Centrifuge 2 min at 700g. Save supernatant for analysis.
10. Repeat Elution 2-4 times. Elution was done 3 times.
Note: Keep the sample chilled as often as possible. After running on SDS-PAGE, adjust the wash steps and elution steps if necessary. Pool any elution fractions with desired sample.
Conclusions: Tomorrow, the SDS-PAGE will be ran in order to assess the purification.
Intro: Digested and ligated Mutant #10
10-fold Digestion:
5 uL buffer 2.1
1 uL Xba
1 uL Spe
1 ug DNA
ddH2O (to 50 uL)
37C 1 hour
80C 15 min (heat inactivation)
Ligation:
2 uL 10x T4 ligase buffer
3:1 molar insert to vector ratio
1 uL T4 ligase
ddH2O (to 20 uL)
16C overnight
65C 15 min (heat inactivation)
Tyler Lee