| Subclone Library Production |
| Version Number: | 1 |
| Start Production Date: | 5/1/99 |
| Author: | Charles Yu, Wade Brannon |
| Edited by: | X |
| Reviewed by: | Amy Geotina |
| Summary |
| Materials & Reagents |
| Materials/Reagents/Equipment | Vendor | Stock Number |
|---|---|---|
| Disposables | ||
| QIAquick 96 PCR Purification Kit | QIAGEN | 28181 |
| LB Amp 100 X-gal Plates (86x128mm) | Teknova | 0116 |
| LB Amp 100 X-gal Plates (254mm) | Teknova | L133-A100X |
| Nunc 96 Well Plate, U Bottom | Applied Scientific | N/A |
| Nunc 384 Well Plate, Cell Culture | Applied Scientific | N/A |
| Chroma Spin + TE-1000 | Clontech | K1324-2 |
| Stock Solutions | ||
| 50mM Tris pH8.0 / 15mM MgCl2 | N/A | N/A |
| 100%Ethanol | N/A | N/A |
| LB Broth with 7.5% Glycerol | Teknova | 0181-G7.5 |
| S.O.C. Medium | Teknova | 0166-10 |
| Reagents | ||
| T4 DNA Polymerase 1U/uL | Boehringer Mannheim | 1004786 |
| DNA Polymerase 1 (Klenow) 5000U/mL | New England Biolabs | 210B |
| Fast-Link DNA Ligation Kit | Epicentre Technologies | LK6201H |
| 2mM dNTP | Amersham | US77170 |
| pUC18 SmaI / BAP 5ug | Amersham | 27-4860-01 |
| MAXEfficiency DH5alpha Competent Cells | GibcoBRL | 18258-012 |
| Equipment | ||
| QIAvac 96 | QIAGEN | 19504 |
| Vaccuum Regulator | QIAGEN | 19530 |
| 2 Bucket Plate Centrifuge | Eppendorf | 5416 |
| 24 Tubes Centrifuge | Eppendorf | 5417 |
| HydroShear | Gene Machines | N/A |
| Hi-Gro | Gene Machines | N/A |
| Procedure |
Shearing:
1. Shear 110uL of 1.5-1.8ug isolated BAC DNA with the HydroShear from Gene Machines. Dilute with sterile filtered ddH2O if necessary.
2. Open the HydroShear program and set the shearing parameters as follows:
a. DNA volume = 110�l, # of Cycles = 20, Speed Code = 15
b. Edit Wash Cycles: 3x with 0.2M HCl, 2x with 0.2M NaOH, 3x with filtered ddH2O.
c. Make sure all wash buffers have been sterile filtered to minimize clogging.
3. After shearing, collect the samples into a 96-well microtiter plate. The volume recovered after shearing should be approximately 100uL. If shearing from multiple HydroShears, array all of the samples into one 96-well microtiter plate.
Filter #1:
1. Assemble the QIAVac vacuum manifold with the waste container inside. Place the QIAquick 96 Plate on top of the manifold.
2. Add 500uL of Buffer PB directly into the wells of the QIAquick 96 Plate.
3. Add the sheared DNA from the microtiter plate to the QIAquick 96 Plate and pipette up and down 20 times to mix thoroughly. Some liquid will start filtering through via gravity flow, but this is normal.
4. Apply a vacuum of 400 mbars to start the filtration process. Turn off the vacuum once all the liquid has filtered through.
5. Add 900uL of Buffer PE to the QIAquick 96 Plate and apply 400 mbars of vacuum pressure to filter the liquid. Make sure 100% ethanol has been added to the stock Buffer PE before using. Turn off the vacuum once all the liquid has filtered through.
6. Add another 900uL of Buffer PE to the QIAquick 96 Plate and apply 400 mbars of vacuum pressure to filter the liquid. Once all the liquid has filtered through, apply maximum vacuum pressure (600 mbars) to remove excess Buffer PE. Apply maximum vacuum for 10 minutes. During the 10 minutes, the vacuum pressure will start decreasing until it levels off at approximately 180 mbars. This is normal.
7. After 10 minutes, turn off the vacuum source. Lift the top plate from the base (not the QIAquick 96 Plate from the top plate), vigorously rap the top plate on a stack of absorbent paper until no drops come out. Then, while holding the plate tightly upside-down, fling the plate 5-8 times quickly to remove excess ethanol. Finally, blot the nozzles of the QIAquick 96 Plate with a clean Kimwipe. Make sure most of the Buffer PE has been removed from the nozzles.
8. For elution into a 96-well microtiter plate:
Replace the waste tray with an empty blue collection microtube rack and place a 96-well microtiter plate directly on the rack. Place the top plate back on the base.
9. To elute, add 60uL of 50mM Tris pH 8.0 / 15mM MgCl2 (warmed to 65oC for 10 minutes) to the center of each well of the QIAquick 96 Plate, let stand for 1 minute, and switch on the vacuum source (400 mbars) for 30 seconds. Make sure all of the liquid completely covers each well before turning on vacuum. After most of the liquid has drawn through, turn up the vacuum to 600 mbars for 5 minutes. Should recover approximately 30-35uL of sample.
Blunt End Repair:
1. Using a multichannel pipette, transfer 32.5uL of sample after Filter #1 to a clean PCR tray.
2. To each sample, add:
4uL 2mM dNTPs
1.8uL T4 DNA Polymerase (1U/uL)
1.7uL Klenow Fragment (5000U/mL)
A cocktail mix of the above (dNTPs, T4 Polymerase, and Klenow) can be prepared and aliquotted to 8-strip PCR tubes so that 7.5uL of the mix can be added to the samples with a multichannel pipette. For example, for a plate of 96 libraries make a cocktail mix of:
| 4uL 2mM dNTPs | X 120 samples= | 480uL 2mM dNTPs |
| 1.8uL T4 DNA Polymerase | X 120 samples= | 216uL T4 DNA Polymerase |
| 1.7uL Klenow Fragment | X 120 samples= | 204uL Klenow Fragment |
| Total= | 900uL | |
|---|---|---|
3. Cap the PCR tray and quick spin down the PCR tray on an Eppendorf 5416 plate centrifuge.
4. Allow the reaction to proceed at room temperature for 30 minutes.
5. After 30 minutes, heat inactivate the PCR tray in a PCR machine with the settings:
70oC for 15 minutes, 4oC forever. The PCR tray can be stored in a 4oC refrigerator overnight.
Filter #2:
1. After heat inactivation, add 200uL of Buffer PB directly into the PCR tray and pipette up and down 10 times to mix. Load all 240uL of samples into a clean QIAquick 96 Plate.
2. Repeats Steps 4-8 from Filter #1.
3. To elute, add 70uL of Buffer EB (warmed to 65oC for 10 minutes) to the center of each well of the QIAquick 96 Plate, let stand for 1 minute, and switch on the vacuum source (400 mbars) for 30 seconds. Make sure Buffer EB completely covers each well before turning on vacuum. After most of the liquid has drawn through, turn up the vacuum to 600 mbars for 5 minutes. Should recover approximately 40-45uL of samples.
4. Proceed to the CHROMA SPIN-1000 Protocol for size excluding small fragments.
CHROMA SPIN-1000 COLUMN PROTOCOL
Preparation:
1. CHROMA SPIN-1000 columns are optimized to handle DNA that is in the 20-1000 ng/uL concentration range and in a sample volume of 70-100uL. Our end-repaired fragments are typically in a 12-24 ng/uL concentration range and in a sample volume of 40-45uL. To make up for the concentration and volume differences, glycogen is used as a carrier. We will increase the concentration of our end-repaired fragments to 50ng/uL in a sample volume of 70uL by adding 25uL of a 116ng/uL glycogen solution.
Purification:
1. Before using a CHROMA SPIN column, it is necessary to invert it several times to resuspend the gel matrix completely.
2. After resuspension, snap off the break-away end of the column and place the column into the 2-mL collection tube provided. Lift off the top cap and place the column/collection tube into the centrifuge.
3. Centrifuge for 5 minutes at 720 x g. After centrifugation, the column matrix will appear semi-dry. This step purges the equilibration buffer from the column and re-establishes the matrix bed.
4. Remove the spin column and collection tube from the centrifuge rotor, and discard the collection tube and equilibration buffer. It is important to handle the spin column gently in order not to disturb the gel matrix.
5. Place the spin column into a clean, labeled 1.5mL microfuge tube. It is not necessary to remove the cap of the tube since the g forces will not cause the caps to break off.
6. Carefully and slowly load the 70uL of end-repaired sample to the CENTER of the gel bed�s slanted surface. Do not allow any sample to flow along the inner wall of the column because this will allow small DNA fragments to filter through, bypassing the gel matrix.
7. Carefully load the spin column/microfuge tube assembly into a centrifuge. It is important at this step orient the spin column the same way it was oriented at the purge stage in step 3. This prevents the gel matrix from breaking apart during the spin. Centrifuge for 1 minute at 720 x g.
8. Remove the spin column and microfuge tube from the rotor and detach them from each other. The purified sample is at the bottom of the microfuge tube. Should recover 60-65 uL.
Agarose Gel Quantification:
1. Take 5uL of purified sample and load into a 1% agarose gel along with sheared mass standards and 1kb ladder.
2. Run the gel for 30 minutes at 120V.
3. Take a photo and quantify the sample.
96-Well Precipitation:
1. While quantifying, in a clean PCR tray add 1uL of stock glycogen (20mg/mL) to each of the wells.
2. Then transfer the remaining samples (~55-60 uL) from the 1.5mL tubes to the corresponding wells of the PCR tray. This can be done more easily with a Matrix expandable multichannel pipette.
3. To each of the wells of the PCR plate (now with sample and glycogen) add:
1/10 V 3.5M NaOAC pH 5.2
2.5 V 100% EtOH
A multichannel pipette can be used to expedite this step.
Cap the PCR plate with the 8-strip caps and invert the PCR tray 20 times to mix the contents. Place the plate in a -20 C freezer for 30 minutes. The plate can be kept in the freezer overnight if necessary.
4. After 30 minutes, centrifuge the plate for 45 minutes at 4,000 rpm in the Eppendorf 5416 plate centrifuge
5. After 45 minutes spin, discard the supernatant by quickly inverting the plate. While keeping the plate inverted, place it on a stack of paper towels to absorb any liquid on the surface of the plate. This will prevent any liquid on the plate�s surface from contaminating adjacent wells once the plate is reverted.
6. Add 200uL of chilled 70% ethanol to each well. Without capping the plate, spin the plate for 10 minutes at 4,000 rpm.
7. After 10 minutes spin, discard the supernatant by quickly inverting the plate and, while keeping the plate inverted, blot any excess liquid on the plate�s surface on paper towels.
8. Place a clean paper towel in the bucket of the plate centrifuge and place the INVERTED plate over the towel. Start the centrifuge. When the speed reaches 300 RPM, STOP the centrifuge and remove the plate. This step should remove excess ethanol in the wells without removing the pellet.
9. Place a clean Kimwipe over the top of the plate and place the plate in the fume hood to air dry for 10-15 minutes.
10. After the pellets have dried, the quatification of the samples should be finished. Resuspend the pellet with enough ddH2O to make the concentration of the DNA to be 20.5ng/uL. By taking 9.75uL of the sample, you should have approximately 200ng for vector ligation.
Vector Ligation:
1. In a clean PCR plate add 1uL of pUC18 SmaI/BAP vector (~100ng) to each well. The pUC18 vector from Amersham/Pharmacia comes lyophilized in 5ug tubes and should be resuspended in 50uL of TE pH 8.0. Make sure to spin down the dry tubes before adding TE for resuspension. Let the vector resuspend for at least 15 minutes before using.
2. Transfer 9.75uL of sample (~200ng) following the precipitation step into the corresponding well of the PCR plate. Quick spin down the plate.
3. In a 1.5mL centrifuge tube on ice, prepare a cocktail mix of:
1.5uL 10X Ligation Buffer (Fast-Link) per sample
0.75uL 10mM ATP (Fast-Link) per sample
2uL Fast-Link DNA Ligase per sample
For 96 libraries on a plate:
| 1.5uL 10X Ligation Buffer | X 120 samples= | 180uL 10X Ligation Buffer |
| 0.75uL 10mM ATP | X 120 samples= | 90uL 10mM ATP |
| 2uL Fast-Link Ligase | X 120 samples= | 240uL Fast-Link Ligase |
| Total= | 510uL | |
|---|---|---|
4. Dispense 4.25uL of cocktail mix to each sample in the PCR plate with a multichannel pipette. Mix up and down 5 times. Cap and quick spin down the plate.
5. Allow the ligation reaction to proceed at room temperature for 1.5 hours.
3. After 1.5 hours, heat inactivate the sample at 70oC for 15 minutes with a hold of 4oC in a PCR machine.
Transformation:
1. In a 1.2mL collection microtube rack (with the outer skirt of the base removed for better heat transfer), add:
1uL Ligation Reaction
50uL of DH5alpha MAXEfficiency Competent Cells
Incubate on ice for 30 minutes. While incubating, prepare a 42oC water bath.
2. After 30 minutes, heat shock the cells in a 42oC water bath for 45 seconds. After heat shock, immediately place the tubes on ice for 2 minutes.
3. After 2 minutes, add 450uL of S.O.C. media to the cells.
4. Place the microtube rack in a 37oC platform shaker set at 200rpm for 50 minutes.
Plating:
1. Before the 50 minutes are up, prepare two LB/Amp/IPTG/X-gal agar plates per library by letting them warm to room temperature.
2. After 50 minutes, plate 200uL of transformation mixture onto one agar plate and 300uL onto the other.
3. Incubate the plates in a 37oC incubator for 20 hours.