Difference between revisions of "Team:UCSC/Experiments"

Designing PCR Primers

Our teaching assistant, McKenna Hicks, taught us the following methods for primer design.

1. Identify the DNA sequence to amplify or stitch together with overlap extension PCR (OE-PCR). During PCR, primers anneal to their complementary sequence and recruit DNA polymerase to extend the sequence in the 5' to 3' Direction.
2. Design a forward (5'- 3') and a reverse primer (3'- 5') to amplify both the top and bottom DNA strands. The ideal primer length is between 18 and 23 bp, but this can be changed to accommodate other constraints.
3. For the forward primer, select a sequence of base pairs (bp) of optimal length.
4. Repeat Step 3 for the reverse primer and generate the reverse complement of the reverse primer sequence.
5. Analyze each sequence using a computational primer tool (such as IDT's Oligo Analyzer) and record the GC content and the melting temperature (Tm) for each primer.
6. The optimal GC content is between 40-60%, and the optimal Tm range is between 52-62℃. Ideally, the difference in Tm for the forward and reverse primer pair should not exceed 5℃.
7. If the GC content exceeds 60% for either primer, add High GC Enhancer to the PCR reaction mix.
8. Check that the free energy (ΔG) value for self-dimers and hetero-dimers on each primer is above -10 kcal/mol to avoid primers spontaneously self-annealing or annealing to each other.
9. Check that hairpin formations do not exceed 30℃ in each primer. PCR uses different temperatures for each thermocycler step; the lowest temperature used during the replication step for primer annealing is 58-60℃. If hairpins form around this temperature, the primer binding efficiency may be reduced and yield less product. However, if the primer Tm is above the hairpin formation temperature, and if the potential hairpin leaves the 3' end of the primer free for annealing, the primer should still anneal properly without reducing PCR Efficiency.

E. Coli Miniprep

The following protocol was adapted from the ZyppyTM Plasmid Miniprep Kit procedure from Zymo Research^[3].

1. First prepare your cell samples: centrifuge the cell cultures (which should be 5 mL in conical tubes) for 15 minutes at 3,750 rpm and 30℃.
2. During waiting time, grab a 250 mL beaker and add roughly 50 mL of 10% bleach. Grab some foil, label \waste", and cover the top of the beaker. Use this beaker to discard biohazardous supernatants and flow-throughs throughout your miniprep.
3. Collect Zyppy columns, Zyppy collection tubes, and 2 sets of 1.5 mL microcentrifuge tubes to match the number of samples you centrifuged. Place the columns over the collection tubes. Be sure to label all vessels accordingly. Place a kimwipe over the open columns.
4. After centrifugation, discard the supernatants into your waste beaker. Be sure to replace the foil lid afterwards.
5. Add 600 L Milli-Q water into each of the conical tubes containing the cell pellets. Resuspend each by slowly pipetting up and down; use that same pipette to transfer the resuspended mixture to its respective clean microcentrifuge tube. Change tips between samples.
6. Add 100 L of 7X Lysis Buffer (blue) and mix by inverting the tube 4-6 times. Proceed to next step within 2 minutes. After addition of 7X Lysis Buffer, the solution should change from opaque to clear blue, indicating complete lysis.
7. Add 350 L of cold Neutralization Buffer and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization.
8. Centrifuge at 13,000 rpm (NOT \rcf" or \x g") for 3 minutes.
9. Transfer the supernatant into the provided Zymo-SpinTM IIN column, which should be on top of a collection tube. Avoid disturbing the cell debris pellet.
10. Centrifuge the column + collection tube for 15 seconds at 13,000 rpm (stop the centrifuge when it reaches 15 sec).
11. Discard the flow-through and place the column back into the same Collection Tube.
12. Add 200 L of Endo-Wash Buffer to the column. Centrifuge for 30 seconds at 13,000 rpm. Discard the flow-through.
13. Add 400 L of ZyppyTM Wash Buffer to the column. Centrifuge for 1 minute at 13,000 rpm. Discard flow-through. Be sure to close cap tightly on wash buffer so that ethanol does not evaporate.
14. Transfer the column into a clean 1.5 mL microcentrifuge tube then add 50 L of Milli-Q water in place of Elution Buffer (EB) and add it as close to the filter as possible, being careful not to touch it (use Milli-Q instead of EB to prevent contamination from salts and EDTA). Let stand for at least 10 minutes at room temperature. Incubate longer if needed.
15. Centrifuge for 30 seconds at 13,000 rpm to elute the plasmid DNA.

Yeast Miniprep

The following protocol was adapted from the ZyppyTM Plasmid Miniprep Kit procedure from Zymo Research^[3].

1. To prepare your cell samples: centrifuge the cell cultures (which should be 5 mL in conical tubes) for 15 minutes at 3,750 rpm and 30C.
2. During waiting time, grab a 250 mL beaker and add roughly 50 mL of 10% bleach. Grab some foil, label \waste", and cover the top of the beaker. Use this beaker to discard biohazardous supernatants and flow-throughs throughout your miniprep.
3. Collect Zyppy columns, Zyppy collection tubes, and 4 sets of 1.5 mL microcentrifuge tubes to match the number of samples you centrifuged (you can get the Zyppy items from the yellow miniprep kit). Place the columns over the collection tubes. Be sure to label all vessels accordingly. Place a kimwipe over the open columns.
4. After centrifugation, discard the supernatants into your waste beaker. Be sure to replace the foil lid afterwards.
5. Add 600 L Milli-Q water into each of the conical tubes containing the cell pellets. Resuspend each by slowly pipetting up and down; use that same pipette to transfer the resuspended mixture to its respective clean microcentrifuge tube. Change tips between samples.
6. Add 100 L of 7X Lysis Buffer (blue) and add 50 L of small glass beads. Vortex for 5 min. Let stand to allow beads to settle.
7. Transfer the supernatant to a fresh microcentrifuge tube.
8. Add 350 L of cold Neutralization Buffer and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization.
9. Centrifuge at 13,000 rpm (NOT \rcf" or \x g") for 10 minutes.
10. Transfer the supernatant into the provided Zymo-SpinTM IIN column, which should be on top of a collection tube. Avoid disturbing the cell debris pellet.
11. Centrifuge the column and collection tube for 45 seconds at 13,000 rpm.
12. Discard the flow-through and place the column back into the same Collection Tube.
13. Add 200 L of Endo-Wash Buffer to the column. Centrifuge for 30 seconds at 13,000 rpm. Discard the flow-through.
14. Add 400 L of ZyppyTM Wash Buffer to the column. Centrifuge for 1 minute at 13,000 rpm. Discard flow-through. Be sure to close cap tightly on wash buffer so that ethanol does not evaporate.
15. Transfer the column into a clean 1.5 mL microcentrifuge tube, then add 50 L of Milli-Q water in place of Elution Buffer (EB) and add it as close to the filter as possible, being careful not to touch it (use Milli-Q instead of EB to prevent contamination from salts and EDTA). Let stand for at least 10 minutes at room temperature. Incubate longer if needed.
16. Centrifuge for 1 minute at 13,000 rpm to elute the plasmid DNA.

Yeast DNA Extraction

We adapted the following procedure from Looke et al.^[4] with advice from our teaching assistant, McKenna Hicks.

1. Add 100L Y. lipolytica in YPD to 100L 200mM LiOAc and 1% SDS solution. Incubate in water bath at 70C for 15 minutes.
2. Add 300L of 96% EtOH. Incubate in freezer overnight.
3. Centrifuge at 8,000 rcf at 4C for 30 minutes. Discard supernatant.
4. To wash pellet: resuspend in 1ml 70% EtOH, incubate for 5 minutes then centrifuge for 10 minutes with the previous speed and temperature conditions. Discard supernatant.
5. Repeat wash step once.
6. Resuspend in 100L 0.5X TE or Milli-Q water; if pellet is not completely resuspended add 0.5X TE or Milli-Q water in 75L increments until completely resuspended.
7. Use NanoDrop to determine concentration and quality of the DNA.

Sourced Procdures

The following is a list of procedures we directly sourced for our project.

• Gibson Cloning^[5]
• Column Electrophoresis via SELEX^[1]
• Random Mutagenesis via Error-prone PCR^[6]
• High Performance Liquid Chromatography and Mass Spectrometry^[7]