Dinh/Dinh 2014/NOTES/2014-5-12
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Producing Stoffel Fragments
- This is the complete protocol to produce Stoffel fragments from E. coli.
- We have most of the reagents in the lab for this protocol.
PCR using the pOpenTaq as template to obtain the Stoffel Fragment DNA & append start codon
- Primer pairs:
Stoffel-F-HindIII AAGCTTATGAGCCCCAAGGCCCTGGAG Stoffel-R-EcoRI GAATTCCTCCTTGGCGGAGAGCCA
- Reaction:
1.5 uL Forward primer (10 uM), Tm=63.1C 1.5 uL Reverse primer (10 uM), Tm=60.0C 1.0 uL pOpenTaq (5ng/uL) 21.0 uL Nuclease-free water 25.0 uL Phusion High Fidelity 2x Master Mix
- Program:
98C (2min) -> [ 98C (30s) -> 55C (30s) -> 72C (60s) ]x30 -> 72C (5min)
- Agarose gel:
File:Stoffel production StoffelPCR.png
Sequential digestion using HindIII/EcoRI on the Stoffel DNA and on pUC19
- Reaction 1: No enzyme control
2.0 uL pUC19/Stoffel (~2ug) 3.0 uL EcoRI buffer (10x) 25.0 uL Nuclease-free water
- Reaction 2: Single HindIII
2.0 uL pUC19/Stoffel (~2ug) 3.0 uL HindIII buffer (10x) 3.0 uL HindIII 22.0 uL Nuclease-free water
- Reaction 3: Single EcoRI
2.0 uL pUC19/Stoffel (~2ug) 3.0 uL EcoRI buffer (10x) 3.0 uL EcoRI 22.0 uL Nuclease-free water
- Incubate each reaction at 37C for 1 hour.
- Reaction 4: Double negative
3.5 uL HindIII buffer (10x) 15.0 uL Reaction 1 33.5 uL Nuclease-free water
- Reaction 5: Double, HindIII then EcoRI
1.5 uL EcoRI 3.5 uL EcoRI buffer (10x) 15.0 uL Reaction 2 30.0 uL Nuclease-free water
- Reaction 6: Double, EcoRI then HindIII
1.5 uL HindIII 3.5 uL HindIII buffer (10x) 15.0 uL Reaction 3 30.0 uL Nuclease-free water
- Incubate reactions 4,5,6 at 37C for 1 hour.
- Run digestion on 1% agarose gel for 1 hour at 50 V. To verify the degree of completion for double digest. Double digestion of pUC19 is required since undigested/single digest vectors will generate colonies under ampicillin selection.
- Purify double digest products using 50% volume of AmPure Beads.
- Agarose gel: (pUC19 digestion only)
- The gel may be slanted since the double digest product is higher than the single-digest product.
File:Stoffel production pUC19 digestion.png
Ligation of Stoffel DNA into pUC19 vector
- Using 50 ng of pUC19 digested products with 94.9 ng of Stoffel DNA digested products.
- Reaction 1: Negative control
12.5 uL Purified digested pUC19 vector (14.3 ng/ul) 12.5 uL Nuclease-free water 2.0 uL Rapid Ligase 25.0 uL Rapid Ligase buffer (2x)
- Reaction 2: Test
12.5 uL Purified digested pUC19 vector (14.3 ng/ul) 12.5 uL Purified digested Stoffel fragment DNA (26.8 ng/ul) 2.0 uL Rapid Ligase 25.0 uL Rapid Ligase buffer (2x)
- Incubate at room temperature for 10 minutes.
Transform ligation product in to Top10 chemically competent cells
- Warm up 3 LB Agar/Ampicillin (100ug/mL) plates in 37C incubator.
NOTE: Blue/white screening can be done at this step by spreading a mixture of 40mg/uL X-Gal and 0.1mM *IPTG onto each plate & let dry before warming up the plates.
- Warm up SOC media in 37C incubator.
- Thaw tubes of competent cells on ice for 10 minutes.
- Add 2 uL of positive control (undigested pUC19) to one tube.
- Add 2 uL of negative control ligation reaction to one tube.
- Add 2 uL of test ligation reaction to one tube.
- Incubate on ice for 30 minutes
- Incubate at 42C (water bath) for 45 seconds.
- Add 250 uL SOC media to each tube.
- Incubate at 37C on ThermoMixer for 1 hour.
- Spread 100 uL of each tube on to separate plates.
- After overnight growth: check that positive control plate have colonies, negative control plate may have colonies if double digestion of pUC19 was incomplete, and there should also be colonies on the test plate.
Colony expansion using transformant colonies
- Select 24 colonies on test plate, and re-streak colonies onto 2 LB/Amp plates (divide each plate into 12 sections & perform dotting using micropipette tips to spread).
NOTE: Blue/white screening can be done at this step by spreading a mixture of 40ug/mL X-Gal and 0.1mM IPTG onto each plate & let dry before warming up the plates.
- After overnight growth: check plate for blue/white colonies if X-Gal/IPTG were used.
- Select one colony from 12 sections & inoculate a 5 mL liquid culture with LB & 100 ug/mL Ampicillin.
- Grow each culture tube with shaking at 225 rpm for at least 6 hours.
Plasmid purification from transformant colonies
- Spin culture tubes for 10 minutes at 4,000 rpm
- Slowly pour off all supernatant
- Add 200 uL of ice-cold GTE (glucose, tris, EDTA) solution to cell pellet and resuspend the cells as much as possible by re-pipetting.
- Add 400 uL SDS/NaOH solution to tube, cap, and invert 5 times. Leave tube at room temperature for 5 minutes.
- Add 300 uL KoAC/Ac solution to tube, cap, and invert 5 times. Leave on ice for 10 minutes.
- Spin tubes at 10,000 rpm for 10 minutes. Pipette ~ 800 uL of supernatant to a new labeled tube.
- Add 500 uL of isopropanol. Leave tube at room temperature for 2 minutes.
- Spin tube for 5 minutes at 10,000 rpm.
- Slowly pour off supernatant. Let tube dry.
- Add 50 uL nuclease-free water and re-pipette to dissolve pellet.
- NOTE: This method of plasmid purification tends to yield small fragments of contaminant DNA, so I used 50% AmPure beads to select for the high molecular weight plasmid DNA. Alternatively, a mini-prep kit can be used to purify plasmids.
Screen plasmids to check for insertion of Stoffel DNA
- Reaction (make a master mix excluding the plasmids & then aliquot):
2.0 uL Forward primer (10 uM) 2.0 uL Reverse primer (10 uM) 16.0 uL Nuclease-free water 25.0 uL Phusion Flash 2x Master Mix 5.0 uL Purified plasmids (diluted to 5-25 ng/uL)
- Program:
98C (10s) -> [ 98C (1s) -> 55C (5s) -> 72C (30s) ] x 30 -> 72C (1min)
- After PCR finished, run all 50 uL of each reaction on a 1% agarose gel for 1 hour at 50 V.
- Agarose gel for screen: Stoffel fragment DNA should be around 1.7 kb.
File:Stoffel production colony screen.png
Production of Stoffel Fragment in E. coli
- NOTE: I performed this step in parallel with Taq (using pOpenTaq) as a positive control.
- Transform a BL21(DE3) competent E coli with positive plasmid from screening step.
- Make an overnight culture of transformed bacteria cells in 5 mL liquid culture with LB and ampicillin (100ug/mL). Culture overnight at 37C (although 30C could be ok).
- Add 1 mL of overnight culture to 100 mL of LB/Amp (100ug/mL).
- The OD of the overnight culture was around OD 3. When the overnight culture is overgrown, the expression culture will grow slowly initially because some of the bacteria in the starter culture loses its plasmid. Grow until OD600=0.4.
- Add IPTG to a concentration of 0.5 mM and grow for 8 hours.
- Harvest bacteria cells by centrifuging at 4,000 rpm for 5 minutes.
- Since Taq & Stoffel should be stable at room temperature, leave the cell pellet at 4C until we are ready to purify the protein.
Purification of Stoffel Fragment from E. coli
- Make 100 mL 4x Taq storage buffer (pH 8.0).
- Note: EDTA will not completely dissolve until the pH is 8.0.
- Measure out appropriate amount of Tris-HCl, DTT, and KCl to add to a single container.
- Add 99 mL of filtered water.
- Pipette appropriate amount of Tween-20, Nonidet-P40 substitute, and 500 mM EDTA to add to solution.
- Use pH paper and 10 N KOH solution to adjust the pH to 8.0.
- Filter the solution and store at -20C.
Reagent | Molecular Weight (g/mol) | Concentration for solutions | Final concentration | Unit | Amount to add | Unit |
Tris-HCl | 157.6 | NA | 20 | mM | 315.2 | mg |
DTT | 154.3 | NA | 10 | mM | 154.3 | mg |
EDTA | 292.2 | 500 mM | 0.1 | mM | 0.02 | mL |
KCl | 74.55 | NA | 100 | mM | 745.5 | mg |
Nonidet-P40 | NA | 100% | 0.5 | % | 0.5 | mL |
Tween 20 | NA | 100% | 0.5 | % | 0.5 | mL |
- Prepare saturated sterile dibasic potassium phosphate by adding 149.25 g of dibasic potassium phosphate to 100 mL of sterile water. Autoclave and UV solution for 30 minutes.
- When cell pellets are ready for protein purification, add 4x Taq storage buffer to 1/20 volume of original culture volumes by vigorous vortex-mixing. If, necessary, transfer the solution to the appropriate container for centrifugation at 16,000 g.
- Incubate in a boiling water bath for 4 minutes, spin at 16,000 g for 10 minutes at 4C, transfer supernatant to a new tube, boil for another 4 minutes, spin at 16,000 g for 10 minutes at 4C, and finally transfer supernatant to a new tube.
- Add sterile dibasic potassium phosphate to the mixture to 10% w/v and mix well for several seconds.
- Add an equal volume of ethanol to the salt solution and mix vigorously for several seconds.
- Spin at 2,000 g for 2 minutes.
- Transfer the organic phase (top layer) to a new tube.
- Add an equal volume of sterilized glyercol and store at -20C.