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Protocols
FUSION PROTEINS AND GENEIOUS
return to topFusion proteins (Using GoldenGate):
Add Q sites using primers.
Make sure to add at least 1 extra nucleotide before the BsaI site (6 is standard). Make sure that the extra nucleotides at the end of the primers are NOT complementary.
Make sure to add/keep the Kozak sequence.
Make sure to get rid of the stop codon of the first protein (all proteins except the last protein).
Make sure the two proteins are in frame.
Check binding site melting temperature at the NEB website. Check hairpin structures in Geneious.
If the BsaI cleavage is scarless, then there should not be a frameshift. (Someone verify this.)
MUTATING DNA SEQUENCES TO GET RID OF CUT SITES
return to topYou are ordering DNA to use with a Golden Gate reaction (using BsaI) to make entry vectors. But... your sequence has BsaI cut sites - oh no! Little fear, you might be able to mutate the codons that make up the BsaI site to remove those sites without changing the amino acid sequence.
Look for BsaI cut sites in your sequence using Geneious. Use Restriction Analysis to find the BsaI sites (under Advanced) and apply them to your sequence.
Select the CDS annotation for your sequence, and check the BsaI sites to see in which codons they occur.
Look for alternate codons that code for the same amino acid. Make sure that the codon that you end up with after mutating has a higher frequency than the existing codon. You can find a table of frequencies for humans here: http://www.genscript.com/cgi-bin/tools/codon_freq_table
If you can't find a codon with a higher frequency, then use a lower frequency codon but you might have to mutate it back (i.e., site-directed mutagenesis*) after doing the Golden Gate reaction.
Here are the changes that we made (4/29/2014):
PirB
VVS: GTG GTC TCC -> GTG GTG TCC
GTC frequency: 0.24
GTG frequency: 0.47
LVS: CTG GTC TCA -> CTG GTG TCA
GTC frequency: 0.24
GTG frequency: 0.47
ET: GAG ACC -> GAA ACC
GAG frequency: 0.58
GAA frequency: 0.42
WARNING: We replaced with a lower frequency codon
LilrB2
VVS: GTG GTC TCC -> GTG GTG TCC
GTC frequency: 0.24
GTG frequency: 0.47
*If you really care, you can mutate these sites back using site-directed mutagenesis (e.g. this kit: http://www.genomics.agilent.com/en/Site-Directed-Mutagenesis/QuikChange-Lightning/?cid=AG-PT-175&tabId=AG-PR-1162)
LOCATING A Q CUT SITE WITHIN A GENE
return to topWhen ordering gBlocks, one must be mindful that the desired DNA does not exceed the maximum size of 2kb. In the case where the gene of interest is close to, or longer than, 2,000 base pairs (for example, the PirB gene is >2,700 base pairs in length), it is necessary to find an appropriate cut site such that 2 (or more) gBlocks may be ordered, and subsequently ligated to reconstruct the whole gene.
The process of identifying and selecting an appropriate cut site within a gene is as follows:
Find the midway mark of the gene. For PirB, this would be around base pairs 1,350 - 1,360.
Identify the 4bp sequence for the Q1 cut site.
Scan the middle region of the gene for this sequence (consider +/- 100bp from the midpoint). For Q1 in PirB, look for 'AGGT' between bases 1,250 to 1,460.
If found, then this is an appropriate cut site: use as the ligation site when constructing pENTR (the entry plasmid).
If not found, repeat the procedure for Q2, then Q3, and so on, until an appropriate cut site is found.
Once an appropriate cut site is located, split the gene into two files, and order each "half" in a separate gBlock.
CHECKING PRIMER TB WITH NEB
return to topLink to Tm Calculator: https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator
Product Group: Phusion
Product: Phusion High-Fidelity PCR Master Mix (HF Buffer)
Primer Conc. (nM): 500
For the primer sequences, input only the binding region and not the entire primer.
For PCR protocol: https://www.neb.com/protocols/2012/09/06/protocol-phusion-high-fidelity-pcr-master-mix-with-hf-buffer-m0531
OLIGOMERIZING BETA AMYLOID
return to topDerived from the Abcam website: http://www.abcam.com/amyloid-beta-peptide-1-42-human-ab120301.html This protocol is for 100 µg polypeptide.
In a fume hood, resuspend 100 µg polypeptide in 100 µL neat HFIP (1,1,1,3,3,3-hexafluoro-2-propanol).
Incubate at room temperature for 1 hour, vortexing briefly every 10 minutes at a moderate speed.
Sonicate in a sonicating water bath on high for 10 minutes.
In a fume hood, place the HFIP/peptide solution under a gentle stream of dry nitrogen gas. Continue until the HFIP evaporates.
Resuspend in 83 µl neat DMSO. Incubate at room temperature for 12 minutes.
Aliquot 10 µl volumes into individual 700 µl microcentrifuge tubes. Store at -80°C.
Oligomerization
Retrieve a single 10 µl aliquot from the -80. Resuspend in 90 µl PBS for 100 µl total solution at a final concentration of 25 µM.
Incubate 2 hours at room temperature to allow for peptide aggregation.
If necessary, remove the insoluble portion by centrifuging at 16,000 xg for 15 minutes.
The solution should be stable at 4° for at least a week.
NOTE: The non-biotinylated ABetan (white tubes, labelled A{Beta}) is in 20 µl aliquots; resuspend in 180 µl PBS for the same working concentration.
As per the LilrB2 paper , fig 1H, LilrB2/Beta amyloid binding gets close to saturation at about 500 nM. This is 10 µl of 25 µM working stock diluted into 500 µl of media (in one well of a 24-well plate.)
Resources (alternate protocols, etc)
Reconstituting Peptide from Package:
http://www.anaspec.com/products/product.asp?id=30304
Add 1% NH4OH directly to powder. Add 70-80 uL NH4OH per 1 mg of the peptide.
Dilute this solution in 1xPBS to your working concentration (should be less than 1mg/mL).
Gently vortex to mix.
Oligomerizing (LilrB2 Paper)
Sonicate for 30 seconds (
Dilute in 1X PBS to a concentration of 100 uM
Incubate at 22C for 16 hours
Incubate at 4C for 24 hours
Centrifuge at 16000 x g for 15 minutes
Collect the supernatant as oligomerized
Oligomerizing (Other protocols)
HFIP Treatment - to ensure monomerization (Abcam protocol)
http://www.abcam.com/amyloid-beta-peptide-1-42-human-ab120301.html
-Dissolve peptide in 100% HFIP. Final concentration = 1mg/mL.
-Incubate at room temperature for 1 hour with occasional vortexing.
-Sonicate for 10 minutes in a water bath sonicator (optional according to some protocols).
-Aliquot into microcentrifuge tubes.
-Dry HFIP-peptide solution under a gentle stream of N2 gas (some protocols leave it to dry in a fume hood - no N2).
-Store at -80C. Do not freeze thaw.
Dissolve an aliquot in 100% DMSO to 5mM.
Add to ice-cold F12 medium (DMEM?) to a concentration of 100 uM.
Incubate at 4C for 24 hours.
Centrifuge at 14000 x g for 10 minutes. Supernatant is a mixture of monomers and oligomers.
This protocol (steps 2-5) is primarily from a paper cited by several other papers, including (indirectly) the LilrB2 paper:
http://onlinelibrary.wiley.com/enhanced/doi/10.1046/j.1471-4159.2001.00592.x/
(HFIP monomerizes http://www.anaspec.com/products/product.asp?id=48257)
Verifying Identity of Supernatant:
Western blot
Math
How much beta amyloid per aliquot?
100 µl aliquot size * 25 µmol/L beta amyloid concentration * (L/10^6 µl) * (1000 nmol / µmol) = 2.5 nmol / aliquot
How many µg is that?
2.5 nmol * (4853.9 g/mol) * (mol/10^9 nmol) * (10^6 µg/g) = 12.1 µg in 10 µL DMSO.
So, 100 µg gets us 8 aliquots of 12.1 ug in 80 µl DMSO
WESTERN BLOT
return to topCell Lysis
For a 24 well plate:
Aspirate media.
Add 500 uL of cold PBS to each well.
Aspirate.
Add 200 uL of trypsin.
Incubate for 90 seconds.
Neutralize with 800 uL of cold complete media.
Pipette up and down to disperse clumps.
Transfer contents of each well to 2 mL labelled eppendorf tubes.
Centrifuge 2500 RPM for 8 minutes.
Aspirate supernatant.
Add 500 uL of lysis buffer.
Incubate at 4C with agitation for 30 minutes.
Centrifuge at 12000 RPM for 20 minutes.
Transfer supernatant to new labelled tubes and store at 4C (or on ice).
Discard pellet.
Denaturing Protein
Add 50uL of betamercaptoethanol (reducing agent) to 950uL of Laemmli dye.
Add Laemmli dye + betamercaptoethanol to lysate so that it dilutes to 1x (equal volumes of Laemmli and lysate if Laemmli is 2x). For one gel, this means that for each sample add 10uL dye to 10uL sample.
Transfer to heat block set to 98C and boil for 5 minutes.
Store at 4C (reboil if reloading).
BCA Assay (to check protein concentration)
For more details: https://www.piercenet.com/instructions/2160412.pdf
For a sample Excel spreadsheet, see: BCR WB BCA 2014-8-1.xls
For a 20mL solution of working reagent, add 10mL Solution A, 9.6mL Solution B, and 0.4mL Solution C to a 50mL tube.
In a 96 well plate, make three rows of protein standards. Add 150uL of protein standard solution and 150uL of working reagent to each well. To make the protein standards:
2. Vial | Volume of Diluent | Volume and Source of BSA | Final BSA Concentration | |
A | 4.5 mL | 0.5 mL of Stock | 200ug/mL | |
B | 8.0 mL | 2.0 mL of vial A dilution | 40ug/mL | |
C | 4.0 mL | 4.0 mL of vial B dilution | 20ug/mL | |
D | 4.0mL | 4.0 mL of vial C dilution | 10ug/mL | |
E | 4.0mL | 4.0 mL of vial D dilution | 5ug/mL | |
F | 4.0mL | 4.0 mL of vial E dilution | 2.5ug/mL | |
G | 4.8mL | 3.2 mL of vial F dilution | 1ug/mL | |
H | 4.0mL | 4.0 mL of vial G dilution | 0.5ug/mL | |
I | 8.0mL | 0 | 0ug/mL (blank) |
STEP | COMPONENT | 6-well | 24-well | 96-well |
1 | Adherent Cells | 0.25-1 x 10^6 | 0.5-2 x 10^5 | 1-4 x 10^4 |
3 | Opti-MEM Medium | 150uL x 4 | 25uL | 25uL x 4 |
Lipofectamine LTX Reagent | 6, 9, 12, 15 uL | 1uL | 1, 1.5, 2, 2.5 uL | |
4 | Opti-MEM Medium | 700uL | 25uL | 125uL |
DNA (0.5-5 ug/uL) | 14ug | 1ug (total) | 2.5ug | |
PLUS Reagent | 14uL | 0.5uL | 2.5uL | |
5 | Diluted DNA (with PLUS) | 150uL | 26uL | 25uL |
Diluted Lipofectamine LTX | 150uL | 26uL | 25uL | |
7 | DNA-lipid complex (per well) | 250uL | 52uL | 10uL |
FINAL DNA (PER WELL)** | 2500ng | 1000ng | 100ng |
SPLITTING CELLS AND SEEDING PLATES
return to top(WELL PLATES SHOULD BE SEEDED ONE DAY PRIOR TO TRANSFECTING)
Prior to transfection, the cells should be 70-90% confluent
Aspirate off the media from the dish the cells are growing in
Add 3mL of PBS/versene, aspirate
Add 2.5 mL of trypsin
Incubate for 2 minutes maximum (do not over trypsinize)
Add 9.5mL of complete media to inhibit the trypsin
Resuspend in a conical tube
Spin down in the centrifuge at 2500 RPM for 5 minutes at room temperature (20-25 C)
Aspirate off the media (be sure not to aspirate the pellet at the bottom!) and resuspend in 10mL of complete media.
Pipette 10-15 uL of culture from the conical tube onto a hemocytometer
Take the hemocytometer to the microscope and count the number of cells in a 4X4 grid (or take an average from multiple 4X4 grids)
The number of cells you count (if you are looking at a large 4X4 grid) is the amount per 0.0001 mL (every 100 cells you count = 1 million cells/ MILLI LITER)
Dilute a sample of the suspension to the correct cell concentration
-A new culture plate needs 1 million cells in 10-12 mL media
-A 24 well plate needs ~50,000 (attractene) or ~100,000 (Lipofectamine) cells in ~0.5mL media per well.
Pipette the necessary volume into each well of your plate for however many wells you need
Tilt/swirl to evenly distribute cells within the wells.
SEEDING PLATES (RAMOS CELLS):
Use modified RPMI-1640 media (it has some added components - the bottle has Brian's initials on it and it's on a shelf in the fridge).
Pipette cells from plate into 50mL conical tube.
Centrifuge at 2500 RPM for 5 minutes at room temperature.
Aspirate off media and resuspend in 10mL Ramos media.
Add 200,000 cells to about 10mL media in a plate (roughly 1:10 split every 3 days).
MAKING CELL CULTURE MEDIA
return to topPrecautions: Make sure that all equipment is functioning as expected first, so that you do not get caught unaware in the middle of a protocol. Media is to be made in the hood. Wash EVERYTHING that goes into the hood. As much as the water is changed in the hot water bath and is blue from an antimicrobial, it is still nasty. We don't want whatever could be in there growing in our culture media, or with our cells.
Materials: DMEM, Fetal Bovine Serum (FBS), Penicilin/Streptomycin (Pen/Strep + L-Glu), Non-Essential Amino Acid
Protocol:
Get a filter column from the cabinet outside the Tissue Culture Room
Attach the vacuum tube to the nozzle
Add 400mL of DMEM to the filter column
Add 50mL of FBS
Add 5mL of 100x Penicillin-Streptomycin-Glutamine
Add 5mL of 100x Non Essential Amino Acids
Let the liquid run through
Add the final 35mL of DMEM to wash through any of the ingredients above through the filter.
PREPARING LB AGAR PLATES
return to top17.5 grams of LB Agar powder makes 500 mL.
1. Weigh 17.5 grams of LB Agar powder, add into sterilized Pyrex container.
2. Fill with sterile water up to 500 mL mark.
3. Cap loosely and tape with autoclave tape.
4. Autoclave for 40 minutes (see an instructor).
5. Wait until cool enough that it doesn't hurt to touch.
If starting from solid agar in the fridge, heat in microwave (cap unscrewed) 3min on high, 5 min on 30% (till it melts completely)
Wait till cool enough so that you can touch it for a minute (60 degree Celsius)
6. Add appropriate volume of Amp or Kan (1000X). For 500 mL of LB Agar, add 500 uL of each antibiotic needed.
7. Swirl to mix, use pipette to transfer 20 mL into each plate. Avoid bubbles.
8. Allow plates to cool and solidify. Mark antibiotic on side of plate.
If LB Agar solidifies before being poured, microwave to liquefy again.
POURING GG DONOR PLATES
return to topStart from solid agar in the fridge or start from powder and follow the protocol.
If starting from solid agar in the fridge, heat in microwave (cap unscrewed) 3min on high, 5 min on 30% (till it melts completely).
Wait till cool enough so that you can touch it for a minute (60 degree Celsius).
Add appropriate volume of Kan (1000X). For 500 mL of LB Agar, add 500 uL of Kan.
Add appropriate volume of X-Gal (1000X). For 500 mL of LB Agar, add 500 uL of X-Gal. (X-Gal is solid at 4 degrees, so take it out to thaw.)
Swirl to mix, use pipette to transfer 20 mL into each plate. Avoid bubbles.
Allow plates to cool and solidify. Mark antibiotic on side of plate.
If LB Agar solidifies before being poured, microwave to liquefy again.
MAKING SOC
return to topS.O.C is made by
dissolving 0.5 ml of 20% glucose in
25 ml of SOB
MAKING CELL STOCKS
return to topAcquire 500 uL of freshly grown cells in liquid culture. This usually comes from the leftover culture that was grown for the miniprep. Fresh Culture can also be grown by inoculating a single colony in TB with appropriate antibiotic marker.
Add 500 uL of culture to microtube.
Add 500 uL of filter sterilized 50% v/v glycerol in water to culture.
Label the tube!
Place in appropriate box in -80C freezer.
For anything important, make two cell stocks (by splitting or by doing the above twice) and store one in the backup cell stock box (Starting 6/28).
ANNEALING AND KINASING OLIGOS
return to top1) Create 100uM stock solutions of each oligo
2) Create a 20uL solution of the following:
2 uL of 100uM stock solution of top oligo
2 uL of 100uM stock solution of complementary bottom oligo
2 uL T4 DNA ligase buffer
1 uL T4 poly nuclease kinase (T4PNK)
13 uL water
3) Put in thermocycler and run the following program:
37°C for 30 mins
95°C for 5 min
Ramp to 25°C by 0.1% (0.1°C per second) and keep at 25°C for 5 min
End at 4°C
BP REACTION
return to top1. Thaw BP clonase on ice
2. Mix 0.5 uL BP clonase with 1 uL appropriate pDONOR (typically ~150 ng/uL) and 1 uL attB-flanked PCR fragment (typically ~50 ng/uL).
3. Incubate at room temperature for 1-1.5 hours.
4. Ad 0.5 uL Proteinase K.
5. Incubate at 37 C 20 min.
6. Transform 1 uL into 10G competent cells.
7. Outgrow w/ SOC 1hr.
8. Plate on Kan, 30C overnight.
TRANSFORMATION
return to topMake sure that the incubator (30/37C) and heat block (42C) are ON.
-Put water in the wells of the 42°C heat block.
Make sure required antibiotic plates are present. Make sure you're using the right antibiotic plates for your plasmid's resistance!
-Warm plates to 37°C. Cold plates reduce transformation efficiency by an order of magnitude.
-Also warm 500 µl SOC per transformation.
Take the DNA out of --20 freezer, let it thaw.
-Vortex DNA to mix, then spin down. Make sure it is completely thawed out!
Make sure that all of the required reagents/DNA etc are present at the site of transformation before you take the cells out of the -80.
Thaw the competent cells on ice for 3-4 min.
-You want to add your DNA right as the last bit of cells' ice melts. Even if it's still a little slushy, that's okay.
Add 1-2 µl of DNA into the comp cells. Stir with a pipette tip a few times, then put right back on ice.
-If you're transforming the result of a reaction (GG, LR, etc) add 1-2 µl of the reaction. Don't add more: many of these reactions have additives that screws up transformation.
-If you're transforming plasmid DNA (from a miniprep), either (a) dilute it out so you add only ~10 ng of DNA, or (b) plate only 10 µl of the outgrowth – else you'll get a lawn! Super-coiled DNA transforms super-efficiently.
Incubate the cells on ice for 30-40 min.
Heat shock the cells for EXACTLY 30 sec at 42 C water bath.
Place back on ice for 2 min.
Add 450 ul of SOC (37° to RT) medium to each tube (S.O.C is made by dissolving 0.5 ml of 20% glucose in 25 ml of SOB. Make sure that the SOC is clear and not cloudy/ contaminated.)
Shake the tubes at 37 C, 280 rpm for 60 min.
Plate 100 µl for a reaction product, or 10 µl in a 100 µl puddle of water for a supercoiled plasmid.
Incubate plates upside down overnight at 37 C or 16-18h at 30C.
Can leave the cells in the incubator for up to 18 hours but no more.
RESTRICTION DIGEST
return to topDigestion Protocol
20 ul total
500-1000 ng DNA
1 ul enzyme
2 ul enzyme buffer
fill the rest with water
Pipette up and down thoroughly
37 degrees for 1-3 hours
4-5 ul loading dye
gel + ladder!
From NEB:
One unit is defined as the amount of enzyme required to digest 1 µg of ? DNA in 1 hour at 37°C in a total reaction volume of 50 µl
So check calculate how much DNA you have and use the right amount of enzyme. Or more.
RESUSPENDING PCR PRIMERS
return to topFrom UTexas protocol
Materials:
TE Buffer
Primer
You will first make a 100 uM master stock
1: spin down primer tube incase there is some primer stuck in cap
2: Calculate amount of buffer needed- multiply nmoles of primer by 10
3: add this many uL of TE to the primer tube
4: gently mix and allow to sit for 10 minutes before making a working stock
PCR
return to topAppending Prefix and Suffix
Things to keep in mind!
Annealing temperature of primers (Tm) should be around 60 C
Check the secondary structure of the primers before you order them!
-no individual secondary structures i.e. hairpins
-no heterostructure with the forward and reverse primers together
-free energy of primers should be greater than -4 kCal
-GC content should be around 50% (40-60% is okay)
If using Phusion Master Mix, use this protocol: https://www.neb.com/protocols/2012/09/06/protocol-phusion-high-fidelity-pcr-master-mix-with-hf-buffer-m0531
Dilute your DNA to the following concentrations:
Template:0.1 - 1 ng/ul
Forward Primer:10 uM
Reverse Primer:10 uM
Set up a small box (e.g. empty pipette tip box) with ice and water. Your DNA and polymerase mix will go into this box before going into the the thermocycler in order to limit endonucelase activity.
Add the following DNA to a labeled 0.6ml PCR tube
DNA:Volume
Template:1 uL
Forward Primer:500 nM
Reverse Primer:500 nM
Program the thermocycler as follows
Temperature:Time
98:30s
PAUSE
98:5s
Tm:15
72:(15s)x(#kb)
72:5m
4:forever
Wait for thermocycler to heat up
Add 22.5uL of polymerase mix (Phusion Master Mix) to your DNA. Mix well and spin down. Transfer tubes to ice as soon as possible.
Once the thermocycler has heated up to the right temperature (it should be paused at 98C), add tubes to thermocycler and resume PCR program.
Calculating Reaction Conditions
Use idtdna.com or VectorNTI to calculate melting temperatures of primers
without common overhangs (base pairs 30 to end when read 5' to 3').
PRIMER
Tm
FW
RV
Phusion elongates at a rate of 1kb (1000bp) per 15s. Look up the length of the
gene of interest and calculate time of elongation.
You should get your Tm from NEB.
If you get the melting temperature of your primer from Genious, the annealing
temperature will be that number minus 2.
Assembling Reaction
Get 0.6mL PCR tubes (not the strip tubes).
Get primers for gene of interest. Resuspend if necessary.
Thaw Phusion supermix on ice.
Add the following (in order):
VOLUME:REAGENT
22.5uL (for 35 cycles):Phusion Supermix
2uL:5uM Forward Primer
2uL:5uM Reverse Primer
1uL:Template DNA(~150ng)
Programming The Thermocycler
(In the 3rd floor thermocycler, the PCR program is named "PHUSION")
Initial Denaturation: 98C for 5min
LOOP: 30-35 cycles
CYCLE:
Denaturation: 98C for 10s
Annealing: calculated temperature (typically 55-65C) for 30s
Elongation: 72C for 15s per kb
Final Elongation: 72C for 10min
Store: 4C
MINI PREP
return to topBefore you Start:
Optional: Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000 and mix (If you're the one adding, initial top and check the box on cap)
Add the provided RNase A solution to Buffer P1, mix, and store at 4C. (One vial of RNase A per bottle of Buffer P1 to give final concentration of 100ug/mL. If you're the one adding, initial top and check box on cap. Buffer P1 will be in the fridge)
Add ethanol (96-100%) to Buffer PE before use (see bottle label for volume) and then check mark on cap.
Check Buffers P2 and N3 for precipitates, if any redissolve by placing in water bath at 37C Do NOT vortex.
Steps
Pellet 1-5mL of bacterial overnight culture by centrifugation at >8000 rpm for 3 min at room temperature (15-25C; use 2ml microcentrifuge collection tubes). Decant all the liquid and add 1 ml of the culture into the corresponding tube. Make sure not to mix up the tries.
Resuspend pelleted bacterial cells in 250 uL Buffer P1 and transfer to microcentrifuge tube.
Add 250uL Buffer P2 and mix thoroughly by inverting tube 4-6 times. Do NOT vortex. Mixture turns blue. Do NOT allow this lysis reaction to proceed for more than 5 min.
Add 350uL of Buffer N3 and mix IMMEDIATELY and thoroughly by inverting tube 4-6 times. Do NOT vortex. Mixture is now colorless.
Centrifuge for 10min at 13,000 rpm in table-top centrifuge.
Apply the supernatant to a QIAprep spin column by decanting or pipetting. Do NOT get any of the sticky precipitate.
Centrifuge for 30 - 60s at 13000rpm. Discard flow-through.
Wash the QIAprep column by adding 0.5 mL Buffer PB.
Centrifuge for 30 - 60s at 13000rpm. Discard flow-through.
Wash the QIAprep column by adding 0.75 mL Buffer PE.
Centrifuge for 30 - 60s at 13000rpm. Discard flow-through.
Centrifuge for 1 min to remove residual was buffer.
Place the QIAprep column in a clean 1.5mL microcentrifuge tube. To elute DNA, add 50uL Buffer EB to center of each column. Be careful NOT to pierce column.
Let stand for 1 minute.
Centrifuge for 60s at 13000rpm.
Remove column and discard, tube now contains DNA.
Go to NanoDrop and spec DNA.
MIDI PREP
return to topNOTES BEFORE STARTING:
Add the provided RNase A solution to Buffer P1, mix, and store at 4C. (One vial of RNase A per bottle of Buffer P1 to give final concentration of 100ug/mL. If you're the one adding, initial top and check box on cap. Buffer P1 will be in the fridge)
Optional: Add LyseBlue reagent to Buffer P2 at a ratio of 1 to 1000 and mix (If you're the one adding, initial top and check the box on cap)
Add ethanol (96-100%) to Buffer PE before use (see bottle label for volume) and then check mark on cap.
Check Buffers P2 and N3 for precipitates, if any redissolve by placing in water bath at 37C. Do NOT vortex.
STEPS:
Harvest bacterial culture by centrifuging at 6000 x g for 15 min at 4C (in 50ml conicals).
Completely resuspend pelleted bacteria in 4ml Buffer P1.
Add 4ml Buffer P2, gently mix by inverting until the lysate appears viscous, and incubate at room temperature (15-25C) for 3 min. If LyseBlue reagent had been added, the cell suspension will turn blue.
Place the QIAfilter Cartridge into a new and suitable tube, allowing space for addition of Buffer BB.
Add 4 ml Buffer S3 to the lysate, and mix by inverting 4-6 times. If LyseBlue reagent has been added, mix the solution until it is completely colorless.
Transfer the lysate to the QIAfilter Cartridge and incubate at room temperature for 10 min. The DNA is now stable; this is an appropriate place in the protocol to pause (if necessary).
During incubation, place QIAGEN plasmid Plus spin columns into the QIAvac 24 Plus. Insert Tube Extenders into each column.
Gently insert the plunger into the QIAfilter Cartridge and filter the cell lysate into the tube.
Add 2 ml Buffer BB to the cleared lysate, and mix by inverting 4-6 times.
Transfer lysate to a QIAGEN Plasmid Plus spin column on the QIAvac 24 plus.
Apply approximately -300 mbar vacuum until the liquid has been drawn through all columns.
To wash DNA, add 0.7 ml Buffer ETR and apply vacuum until the liquid has been drawn through all columns.
To further wash the DNA, add 0.7 ml Buffer PE and apply vacuum until the liquid has been drawn through all columns.
To completely remove the residual wash buffer, centrifuge the column at 10,000 x g (9,700 rpm) for 1 min in a tabletop microcentrifuge.
Place the QIAGEN Plasmid Plus spin column into a clean 1.5 ml tube. To elute the DNA, add 200 ul Buffer EB or water to the center of the QIAGEN Plasmid Plus spin column, let it stand for > 1 min, and centrifuge for 1 min.
MAKING LIQUID CULTURE
return to topMINI-PREP:
Prepare culture in a 15mL, round bottom tube.
Add 3-5mL LB
Add 3-5uL (respectively) of antibiotic (Ampicillin, Kanamycin, etc.)
Pick colony using a pipette tip. Eject tip into tube (tip should remain in tube).
MIDI-PREP:
Prepare culture in a 500mL Erlenmeyer flask.
Add 50mL LB
Add 50uL of antibiotic (Ampicillin, Kanamycin, etc.)
Pick colony using a pipette tip. Eject tip into tube (tip should remain in tube).
LR GATEWAY
return to topPro Tips
Run at the larger scale
ALWAYS kill it with PRoteinase K
Transform 4 uL of the reaction
Use ALL the transformation tricks
Plate ALL of it
ALWAYS run a PUC19 control for transformations
Instructions for newbies:
1.5 uL of 10 fM Dest
1.5 uL of 5 fM promoter
1.5 uL of 5 fM gene
1.5 uL of H2O
1.5 uL of LR clonase
Pipette up and down. Incubate at room temperature overnight.
Next day: Add 1.5 uL of proteinase K. Incubate for 15 minutes at 37C, then transform.
Instructions:
USE 3x VOLUME OF EVERYTHING (at least for now)
Use nanodrop to measure concentration of pEntry vectors, make 5 femtomolar working solution of each pEntry.
Excel sheet set up to calculate the necessary volumes
LR (concentration) calculations.xlsx
Sample calculation:Combine into 1 aliquot:
1uL of 5 fmol of pENTR_L4_Promoter_R1
1uL of 5 fmol pENTR_L1_Gene_L2
1uL of 10 fmol pDEST_R4_R2
3uL Total
WARNING: KEEP ALIQUOTED LR CLONASE MIX AT -80 AT ALL TIMES!
Add 0.5uL LR Clonase Enzyme
Additionally, remember to mix your reactions well after all elements have been added (use a 10 uL pipette set to 3 or 4 uL, then just pipette up and down gently)
Leave at room temperature for minimum of 16 hours, maximum of 24 hours.
transform just 1 uL of the reaction (This should result in around 50-500 colonies (more often than not closer to 500) with high (~90-95%) efficiency)
or storage -20C freezer.
GOLDEN GATE
return to top50 ng of each piece of DNA being joined
Use nanodrop to find concentration in ng/ul, then divide 50 by that concentration to find the required volume of DNA:
Conc: x ng/uL
Vol: 50/x uL
NOTE: If GGDonr is too concentrated, dilute it with EB or water.
NOTE: Ligase buffer does not like to be freeze-thawed, so use one-time-use aliquots.
x1 uL of DNA1
x2 uL of DNA2
y uL (100ng) Donor
2ul 10X T4 Ligase Buffer
2ul 10X BSA
1ul BsaI (enzyme) HC (high concentration)
1ul T4 Ligase (enzyme) HC (high concentration)
fill to 20uL with SDIH20 (put water in before the buffer and enzymes)
-------------
20ul total
(NOTE: Make sure that Buffer and Enzyme added last, enzyme after buffer)
Take a p20, set it to 10uL and then pipet up and down.
THERMOCYCLER:
(Protocol EBGG)
37C for 5min
Part 1
50X:
37C for 2.5min
4C for 0.5min
16C for 5.5min
Part 2
37C for 10 min
80C for 20 min
4C hold (for 8+ hours)
(Check protocol by looking up the paper or other online GG protocols)