Peptide Synthesis: Difference between revisions
PreDec2022>Acastellan45 No edit summary |
PreDec2022>Acastellan45 No edit summary |
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2. While warming, set up clean cartridges in a metal rack. To reduce deletion sequences and improve peptide quality, make sure to double couple each peptide. | 2. While warming, set up clean cartridges in a metal rack. To reduce deletion sequences and improve peptide quality, make sure to double couple each peptide. | ||
3. Weigh out each amino acid (A.A) | 3. Weigh out the amount of each amino acid (A.A) required for a 0.5 mmol reaction. Pour into labeled cartridge. Do NOT create a cartridge for the A.A on the C-terminal end of the peptide. | ||
4. Parafilm cartridges and place back into the fridge. | 4. Parafilm cartridges and place back into the fridge. | ||
* Time permitting, skip step 4 and | * Time permitting, skip step 4 and move directly to the ''Day 2 Protocol''. | ||
'''Day 2: HBTU and Synthesis''' | '''Day 2: HBTU and Synthesis''' | ||
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1. Remove metal racks from the fridge and allow them to warm at room temperature. | 1. Remove metal racks from the fridge and allow them to warm at room temperature. | ||
2. Weigh out | 2. Weigh out the amount of HBTU required for 0.5 mmol reaction. Place into each cartridge. | ||
3. Cap each cartridge with a metal cap and black rubber stopper using the clamp tool. | 3. Cap each cartridge with a metal cap and black rubber stopper using the clamp tool. | ||
4. | 4. Add 0.1 mmol resin to the reaction vessel. ''Resin varies between peptide samples. Different resins are used because they are attached to the A.A on the C-terminal end of the peptide''. | ||
5. Run peptide synthesis on the Gryos 2-Channel Peptide Synthesizer. To operate the machine: | |||
a. Select ''Load Synthesis'' on the peptide synthesizer to begin loading Reaction Vessel 1. | |||
b. Choose first program: 0.1mmol_DC_1_swell_20min. (Remember, all peptides are loaded from the C-terminal to the N-terminal end. Thus, the first sample will be the cartridge containing the resin linked to most the C-terminal A.A. Additionally, the swell program is ONLY used on the resin sample) | |||
c. Load A.A and select forward. | |||
d. Repeat with cycle number 2: 0.1 mol_DC_20min. | |||
e. Continue until all A.A are loaded | |||
f. If running two peptide syntheses, use manual operations to move to Reaction Vessel 2. | |||
g. Repeat steps 5b - 5e. | |||
* MAKE SURE KARIN IS IN THE ROOM TO ASSIST DURING THIS STEP. Remember, this is her equipment and she is eager to help operate the machine and load all of the solvents needed to run the reactions. | |||
Revision as of 20:56, 5 April 2019
Day 1: Weighing Out Peptides
1. Take A.A from the fridge to let them warm at room temperature (This prevents water from condensing into containers which may disrupt the reaction).
2. While warming, set up clean cartridges in a metal rack. To reduce deletion sequences and improve peptide quality, make sure to double couple each peptide.
3. Weigh out the amount of each amino acid (A.A) required for a 0.5 mmol reaction. Pour into labeled cartridge. Do NOT create a cartridge for the A.A on the C-terminal end of the peptide.
4. Parafilm cartridges and place back into the fridge.
- Time permitting, skip step 4 and move directly to the Day 2 Protocol.
Day 2: HBTU and Synthesis
1. Remove metal racks from the fridge and allow them to warm at room temperature.
2. Weigh out the amount of HBTU required for 0.5 mmol reaction. Place into each cartridge.
3. Cap each cartridge with a metal cap and black rubber stopper using the clamp tool.
4. Add 0.1 mmol resin to the reaction vessel. Resin varies between peptide samples. Different resins are used because they are attached to the A.A on the C-terminal end of the peptide.
5. Run peptide synthesis on the Gryos 2-Channel Peptide Synthesizer. To operate the machine:
a. Select Load Synthesis on the peptide synthesizer to begin loading Reaction Vessel 1.
b. Choose first program: 0.1mmol_DC_1_swell_20min. (Remember, all peptides are loaded from the C-terminal to the N-terminal end. Thus, the first sample will be the cartridge containing the resin linked to most the C-terminal A.A. Additionally, the swell program is ONLY used on the resin sample)
c. Load A.A and select forward.
d. Repeat with cycle number 2: 0.1 mol_DC_20min.
e. Continue until all A.A are loaded
f. If running two peptide syntheses, use manual operations to move to Reaction Vessel 2.
g. Repeat steps 5b - 5e.
- MAKE SURE KARIN IS IN THE ROOM TO ASSIST DURING THIS STEP. Remember, this is her equipment and she is eager to help operate the machine and load all of the solvents needed to run the reactions.