Team:LASATX/Experiments
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Protocols
STEP 1: PCR amplification (frag into cloning vector)
Reactants
| Hotstart Accuprime pfx mastermix | 45 uL |
| DNA template | X uL (30 ng) |
| primers (20 uM) | 2 uL |
| MgCl2 (25 mM) | 2 uL (1 mM) |
| Total Volume | 49 + X uL |
Thermocycler (touchdown)
95 C 5 min (2 min if NOT using Hotstart)
// 10 cycles //
95 C 20 s
0.3 C/s to 50 C
72 C X s (extension = 1 min/kb)
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// 15 cycles //
95 C 20 s
55 C 20 s
72 C 1 min
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72 C 10 min
4 C forever
STEP 2A: Gel Electrophoresis
Making Gels
1% TAE
Note
Use large combs for 50 uL PCR reactions and small combs for 10 uL reactions
1. 100 mL TAE buffer + 1 g agarose (Nuseive 3:1)
2. Stir
3. Microwave
4. Add 1 drop of EtBr
5. Let the gel cool
Gel Electrophoresis
Run PCR product through gels
1 kb ladder (2 uL), loading dye (2uL), water (8 uL)
pcr product (45 uL), dye (7.5 uL) (6:1 ratio)
STEP 3: Gel Purification (Promega)
Gel Dissolving the Gel Slice
1. Load and run the dye
2. Weight a 1.5 mL microcentrifuge tube for each DNA fragment to be isolated and record weight.
3. Visualize and photograph the DNA using a long-wavelength UV lamp and an intercalating dye (EtBr). Excise DNA fragment of interest in a minimal volume of agarose using a clean scalpel or razor blade. Transfer the gel slice to the weighed microcentrifuge tube and record the weight. Subtract the weight of the empty tube from the total weight to obtain the weight of the gel slice
Note: The gel slice may be stored at 4°C or at –20°C for up to one week in a tightly closed tube under nuclease-free conditions before purification.
4. Add Membrane Binding Solution at a ratio of 1 mL of solution per 1 g of agarose gel slice.
5. Vortex the mixture and incubate at 50–65°C for 10 minutes or until the gel slice is completely dissolved. Vortex the tube every few minutes to increase the rate of agarose gel melting. Centrifuge the tube briefly at room temperature to ensure the contents are at the bottom of the tube. Once the agarose gel is melted, the gel will not resolidify at room temperature.
DNA Purification by Centrifugation
1. 1. Place one SV Minicolumn in a Collection Tube for each dissolved gel slice or PCR amplification.
2. Transfer the dissolved gel mixture or prepared PCR product to the SV Minicolumn assembly and incubate for 1 minute at RT
3. Centrifuge 1 minute, and discard liquid
4. Wash with 700µl of Membrane Wash Solution, centrifuge 1 minute, discard.
5. Wash with 500µl of Membrane Wash Solution, centrifuge 5 minutes, discard.
6. Centrifuge 1 minute with the microcentrifuge lid open (or off ) to allow evaporation of any residual ethanol.
7. Transfer SV Minicolumn to a clean 1.5ml microcentrifuge tube.
8. Apply 15 µl of Nuclease-Free Water directly to the center of the column. Make sure membrane is completely covered with Nuclease-free water. Incubate at RT for 1 minute. Centrifuge 1 minute at 16,000 × g. Repeat.
9. Discard SV Minicolumn, store tube containing the eluted DNA at 4°C or –20°C.
Notes for "DNA Purification by Centrifugation"
MWS = Membrane Wash Solution
RT = room temperature
Centrifuge at 16,000 x g
Nanodrop Speed/Vac
If concentration not high enough, speed vac and spin again
STEP 4: PCR Clean-Up (Zymo) of Remaining PCR Product
Protocol
All centrifugation steps should be performed between 10,000 - 16,000 x g
1. In a 1.5 ml microcentrifuge tube, add 2-7 volumes of DNA Binding Buffer to each volume of DNA sample (see table below). Mix briefly by vortexing.
2. Transfer mixture to a provided Zymo-Spin™ Column in a Collection Tube.
3. Centrifuge for 30 seconds. Discard the flow-through.
4. Add 200 µl DNA Wash Buffer to the column. Centrifuge for 30 seconds. Repeat the wash step. 5. Add 7 uL water directly to the column matrix and incubate at room temperature for one minute. Transfer the column to a 1.5 ml microcentrifuge tube and centrifuge for 30 seconds to elute the DNA.
Ultra-pure DNA is now ready for use
STEP 5: Gibson
Reactants
gibson mix1, 1.3 X 15 uL
insert2 (~20 ng/uL) 2.5 uL (50 ng)
vector pcr 2.13 2.5 uL
Total Vol 20 uL
Protocol
Incubate 1 hour at 50 C in thermocycler.
1. 15 uL aliquots in pcr tube box OR box next to antibiotics in B lab freezer
2. pcr product (gel purified or pcr clean-up’d)
3. blue sticker
STEP 6A: Chemical Transformation: One Shot TOP10 Competent Cells
Chemical Transformation
1. place Gibson reaction on ice
2. Thaw on ice one 50 uL vial of One Shot TOP10 cells* for each ligation/transformation
3. Pipette 5 uL of each Gibson reaction (OR 1 uL of miniprep) directly into the vial of competent cells and mix by tapping gently. Do not mix by pipetting up and down. The remaining Gibson mixture(s) can be stored at -20 C.
4. Incubate vials on ice for 30 minutes.
~take out vial of SOC~
5. Incubate for exactly 30 seconds in the 42C water bath. Do not mix or shake.
6. Remove vial(s) from 42C bath and place them on ice.
7. Add 250 uL of pre-warmed SOC medium to each vial. SOC is a rich medium; sterile technique must be practiced to avoid contamination.
8. Place vials in microcentrifuge rack on its side and secure with tape to avoid loss of the vial(s). Shake the vial(s) at 37 C for exactly 1 hour at 225 rpm in a shaking incubator. ~take out agar plates. place at 37 C~
9. Spin down and resuspend in 100 uL LB.
10. Spread 50 uL from each transformation vial on separate, labeled LB agar plates. Store the remaining transformation mix at 4C.
11. Invert the plates and incubate at 37 C overnight.
12. Select colonies and analyze by plasmid isolation, pcr or sequencing.
*B lab freezer, bottom row, right most, white pcr tube box
STEP 6B: Electrotransformation of E. coli
electroporate: pulse at 2.5 kV
STEP 7: ON Cultures
Per Culture Tube:
Toothpick w/ Colony
5 mL LB
5 uL Antibiotic
STEP 8: Making Glycerol Stocks
1. In a microcentrifuge tube, add:
a. 800 uL ON culture
b. 200 uL 80% glycerol
2. Freeze at ~80 C
STEP 9: Miniprep (Qiagen)
Procedure
1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 (w/ RNase A*) and transfer to a microcentrifuge tube. No cell clumps should be visible after resuspension of the pellet.
2. Add 250 µl Buffer P2 and gently invert the tube 4–6 times to mix. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
3. Add 350 µl Buffer N3 and invert the tube immediately but gently 4–6 times. To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet will form.
5. Apply the supernatants from step 4 to the QIAprep Spin Column by decanting or pipetting.
6. Centrifuge for 30–60 s. Discard the flow-through.
7. (Optional): Wash the QIAprep Spin Column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through. This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5α™ do not require this additional wash step.
8. Wash QIAprep Spin Column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
9. Discard flow-through; centrifuge 1 min to remove residual wash buffer.
10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube.
11. To elute DNA, add 25 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep Spin Column, let stand for 1 min, and centrifuge for 1 min. Repeat for 2x DNA.
*want 50 ug/mL RNase A in P1
STEP 10: Sequencing Sample Submission
1. Add DNA to a total of 250 ng
2. Use 10 pmol primer (1 uL of 10 uM) per sample
3. Add water to 12 uL
4. Send individual samples in 1.5 mL tubes
5. Write order number on top of each tube and label
6. Spin down
7. Place in green racks in fridge next to room 1.42G
Note: Samples put in before 10 am will be sequenced