Protein A FLAG Tag M10018 M10019: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
John Yi Wang (talk | contribs) No edit summary |
John Yi Wang (talk | contribs) No edit summary |
||
| Line 16: | Line 16: | ||
*For our 100nm stock: | *For our 100nm stock: | ||
29 uL water | 29 uL water | ||
5 uL Expand 10x Buffer 2 | 5 uL Expand 10x Buffer 2 | ||
5 uL 10x dNTPs (2 mM in each; 0.2 mM final conc) | 5 uL 10x dNTPs (2 mM in each; 0.2 mM final conc) | ||
| Line 31: | Line 31: | ||
(or something similar) | (or something similar) | ||
'''Protip''' | '''Protip'''<br> | ||
No gel is required because your pieces are too small to see. | No gel is required because your pieces are too small to see.<br> | ||
Your next step is "special" small frag cleanup as oppose to the large frag. This removes the dna polymerase. | Your next step is "special" small frag cleanup as oppose to the large frag. This removes the dna polymerase. | ||
| Line 38: | Line 38: | ||
== '''Small-Frag Zymo Cleanup''' == | == '''Small-Frag Zymo Cleanup''' == | ||
The following procedure removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction for fragments smaller than 300bp. It also will remove the buffer and restriction enzymes from a restriction digest reaction | *The following procedure removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction for fragments smaller than 300bp. It also will remove the buffer and restriction enzymes from a restriction digest reaction: | ||
1. Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction. | 1. Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction. | ||
2. Transfer into the Zymo column (small clear guys) | 2. Transfer into the Zymo column (small clear guys) | ||
| Line 49: | Line 48: | ||
8. spin through, discard waste. | 8. spin through, discard waste. | ||
9. spin for 90 seconds, full speed to dry. | 9. spin for 90 seconds, full speed to dry. | ||
10. elute with water into a fresh Eppendorf tube | 10. elute with water into a fresh Eppendorf tube | ||
== '''EcoRI/BamHI Digest of Wobble Products''' == | == '''EcoRI/BamHI Digest of Wobble Products''' == | ||
Revision as of 19:37, 16 February 2009
Protocol for making Protein A FLAG Tag M10018 M10019
GOOD LUCK!
Oligos
- The initial oligos will be at 28.8 nanoM. You want a conc. of 100nM give or take. - Add 288ul of water. This makes the conc. 100nM of the oligos Protip - Spin down oligos before adding water to make sure it is at the bottom. due the crappy spinner, no balancing required. - Mix (by scrapping it against the tube rack ---> high tech!) then spin down again.
Wobble
- For our 100nm stock:
29 uL water 5 uL Expand 10x Buffer 2 5 uL 10x dNTPs (2 mM in each; 0.2 mM final conc) 5 uL Oligo 1 (100uM) 5 uL Oligo 2 (100uM) 0.75 uL Expand Polymerase 1
- The wobble program should be preset. Just in case, here's the settings:
2 min at 94 10 cycles of: 30 sec at 55 30 sec at 72 (or something similar)
Protip
No gel is required because your pieces are too small to see.
Your next step is "special" small frag cleanup as oppose to the large frag. This removes the dna polymerase.
Small-Frag Zymo Cleanup
- The following procedure removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction for fragments smaller than 300bp. It also will remove the buffer and restriction enzymes from a restriction digest reaction:
1. Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction. 2. Transfer into the Zymo column (small clear guys) 3. Add 500uL of Ethanol and pipette up and down to mix 4. spin through, discard waste. 5. Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol) 6. spin through, discard waste. 7. Add 200 uL of PE or Zymo Wash buffer 8. spin through, discard waste. 9. spin for 90 seconds, full speed to dry. 10. elute with water into a fresh Eppendorf tube
EcoRI/BamHI Digest of Wobble Products
For wobble products, you will digest the entire extension reaction-worth of DNA Since you started with 50uL of extension reaction, you should now have 50uL of eluted DNA in water.
- Set up the following reaction in a PCR tube:
50uL eluted DNA
5.7uL NEB Buffer 2 1uL EcoRI 1uL BamHI
* Mix thoroughly by slamming the tube upside down on the table, and then a quick spin to move the liquid to the bottom of the tube.
* Incubate the reaction at 37 degrees on the thermocycler
* Proceed to another Zymo small fragment cleanup
