Protein Secondary Structure predictions online

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https://molbiol-tools.ca/Protein_secondary_structure.htm

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Calcium Phosphate transfection for Lentivirus production

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Calcium Phosphate transfection for Lentivirus production

DNA

  • 80 µg of DNA per T175 (or 150 mm dish)
    • 40 µg of lentiviral vector
    • 32 µg of psPAx2
    • 8 µg of pMD2.G
  • Or 80 µg of any other lentiviral vector

Solutions

2X HEPES solution (Preparing in advance, test and freeze aliquot)

  • 280 mM NaCl
  • 50 mM HEPES free acid
  • 5 mM Na2HPO4
  • Adjust pH between 6.9 to 7.2 (our best result is at 7.05)
    • The pH of this solution is critical. You can test different pH in this range and freeze aliquot of the best one. Do not put in the fridge or a freezer door, the pH can change with time.

2.5 M CaCl2

  1. Dissolve 2.5 M of CaCl2
  2. Filter sterilize at 0.2 µm
  3. Keep this solution in the fridge or aliquot and put at -20°C

1 M Sodium Butyrate (Butyric acid sodium salt) stock solution

  1. Filter sterilize at 0.2 µm
  2. This solution can be keep in the fridge
  3. 1000X stock solution

Protocol

Day 1

  1. Plate 25 million of HEK293T cells (mycoplasma free, very important) per T175 (or 150 mm dish) poly-L-ornithine (can also be poly-D-lysine or poly-L-lysine) coated in 20 ml of FBS 10% or BCS 10%
    1. BCS (bovine calf serum) is a lot cheaper than FBS and do the same job
    2. Confluence at 60% next morning

Day 2

  1. Change medium for 15 ml of DMEM FBS 2.5% (or BCS 2.5%).
  2. Put the cell in incubator for at least 30 min before transfection.
  3. In a 15 ml tube (mix A)
    1. Lentiviral vector
    2. psPAx2
    3. G
    4. 150 µl of 2.5 M CaCl2
    5. Complete volume at 1.5 ml with ddH2O
  4. Bubble air the mix A adding 1.5 ml of 2X HEPES (room temperature) in the tube (dropwise)
    1. Critical step: While bubbling hard with a pipette gun, adding the 2X HEPES slowly drop by drop. You should see the solution turning cloudy. Bubbling is the key to the success (with the 2X hepes pH). The more intense is your bubbling, the smaller is your DNA/CaCl2 particles and the better is your transfection.
  5. Vortex
  6. Incubate 30 min at RT
  7. Vortex
  8. Add DNA/HEPES mix dropwise in the flask while mixing
  9. Incubate O/N

Day 3 (18-21h post-transfection)

  1. Change medium for DMEM 10% FBS (16 ml)
    1. Bleach the discarded medium
  2. Add 16 µl of 1M sodium butyrate
  3. Incubate O/N

Day 4

  1. Harvest virus and add 15 ml of medium in each dish
  2. Incubate O/N
  3. Centrifuge the harvested virus at 2000g for 10 min at 4 °C
  4. Filter the supernatant with 0.45 µm filter (or bigger, not smaller)
  5. Keep the harvested virus at 4 °C O/N or go directly to virus concentration

Day 5

  1. Harvest virus and bleach the dish
  2. Centrifuge the harvested virus at 2000g for 10 min at 4 °C
  3. Filter the supernatant with 0.45 µm filter (or bigger, not smaller)
  4. Virus concentration

Virus ultracentrifugation concentration

Solutions

20% sucrose solution

  1. Dissolve 20 g of UltraPure sucrose
  2. 100 mM NaCl
  3. 20 mM HEPES (pH 7.4)
  4. 1 mM EDTA
  5. Adjust the volume to 100 ml by adding H2O and filter-sterilize. This solution can be stored at 4°C for at least 6 months.

Protocol

  1. Filter your virus
  2. Add 30 ml (or less) of the unconcentrated virus solution into centrifuges tubes
  3. Add 4 ml of 20% sucrose under the lentivirus solution
    1. Be careful to not mix the virus solution with the sucrose.
  4. Weight each of your tubes and balanced them to the exact same weight
  5. Ultracentrifuge for 2 hrs at 82,700g (25K rpm Beckman SW28) at 4°C
  6. Remove and bleach the supernatant and leave the tubes up-side down on paper towel for few min to remove the residual liquid.
  7. Wipe out the residual liquid on the edge of the tube.
  8. The pellet is normally invisible (if you saw one, it is probably cell debris)
  9. Add 300 µl of DMEM or PBS (for a concentration of 100X) and incubate at 4°C for 2 hrs. Pipette up and down without creating bubble every 20-30 min (it’s hard to resuspend, some authors let the virus at 4°C O/N)
  10. Transfer the concentrated virus in cryogenic tubes, snap freeze and store the stock at -80°C

Lentivirus quantification

The titration of lentivirus is important to troubleshoot transfection problems and to produce reproducible transduction, specially for hard to transduce cells. If the lentiviral construct is conjugated with a fluorescent protein, this titration can be done by Flow Cytometry or by High content microscopy. If your lentivirus construct doesn’t have a fluorescent protein, the titration need to be done by qPCR. A protocol for lentivirus titration will be produce on demand.

Lentivirus transduction tips

Solution

8 mM Hexadimethrine bromide (polybrene) stock solution

  1. Filter sterilize at 0.2 µm
  2. This solution can be keep in the fridge
  3. 1000X stock solution

Tips

  1. Determine a good MOI (multiplicity of infection) for your cell type.
  2. The transduction should be done in a minimum serum (better without serum) for at least 6h.
  3. Adding 8 µM of polybrene can improve lentivirus transduction by 10 times
  4. Light centrifugation of the plate (300g) with the lentivirus can improve lentivirus transduction by 10 times and probably can reduce the transduction incubation.

 

==============

Tips for lentiviral transduction

DON’T:

  1. Don’t expose to environmental extremes (such as pH, temperature, organic solvents, protein denaturants, strong detergents, or cation chelators such as EDTA).  ALLliquids the virus is in (dilution buffers, cell culture media, etc.) should be pH= ~7.2.  Add 10 mm HEPES if in doubt to buffer the pH.
  2. In particular the VSV-G protein, which is on the surface of the virus and is required for infection, is extremely pH sensitive.  It loses about 10-fold in infectious activity for each 0.3 unit drop in pH below ~pH 7.0.  Although this effect is reversible to some degree the reversibility is time dependent.  Because of this pH sensitivity ALL liquids the virus is in (dilution buffers , cell culture media, etc.) should be pH= ~7.2.  Add 10 mM HEPES if in doubt to buffer the pH.
  3. Don’t expose to any condition that disrupts membranes (such as temperature, organic solvents, and strong detergents).  This will result in near complete inactivation because an intact viral membrane is required for viral infection.
  4. Don’t introduce air into the virusby vortexing, blowing bubbles and similar operations which result in protein denaturation.  Denatured VSV-G, which is required for infection, is inactive.
  5. Don’t dry. Drying will also result in membrane disruption and near complete inactivation of the virus.
  6. Don’t freeze and thawmultiple times.  The titer may drop 2-3 fold (or more) with each freeze-thaw cycle.   Therefore, it is best to avoid.
  7. Don’t expose to hydrophobic plastics(especially polystyrene) for prolonged periods.  Because lentiviruses are surrounded by a mostly hydrophobic membrane they are very sticky and losses can occur if they are exposed to hydrophobic plastics while not frozen.  It is best to store thawed lentiviruses in siliconized or low protein binding tubes and pipette it with similar pipette tips.
  8. Don’t filter. Filtering can reduce titer because viruses can stick to the filter.  If filtering is necessary it is essential that filters with 0.45 μm (or larger) pores are used.  Since the diameter of a lentivirus is about 0.15-0.2 μm, if a filter with 0.2 μm (or smaller) pores is used substantial loss of infectious titer can occur.

DO:

  1. Do aliquot and freezeat -80C for long term storage if the virus is not used within ~1 week.  Besides being intrinsically unstable it is possible for microorganisms to grow in residual cell culture media that is in the virus.  Freezing helps prevent this.
  2. Thawon ice just before use.
  3. Usevirus within ~ 6 months of storage at -80C unless the titer is much higher than needed.  Even when stored at -80C lentiviruses will lose ~10x in titer every 6-12 months.

=================

Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors     Published online 19 March 2009; doi:10.1038/nprot.2009.22
https://www.nature.com/nprot/journal/v4/n4/pdf/nprot.2009.22.pdf?origin=ppub

 

Making Competent Bacterial Cells for Transformation

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http://molecularbiology.forums.biotechniques.com/viewtopic.php?p=71711

PEG/DMSO transformation (which I’m a fan of, heheh), it’s very easy. The protocol is based on Chung and Miller, 1988.

To prepare competent cells, just grow E. Coli to OD ~0.5, pellet them by gentle centrifugation at 4°C, resuspend in 1/10th of the original culture volume of ice-cold, sterile-filtered TSB buffer (LB pH 6.1 + 10% PEG-3350 + 10 mM MgCl2 + 10 mM MgSO4 + 5% DMSO) and incubate for 10 minutes on ice. Store in 250 or 350 ul aliquots at -80°C.

My lab’s protocol is slightly different from Chung and Miller’s in the transformation part. We add in a chilled eppendorf 20 ul of 5xKCM buffer (0.5M KCl, 0.15M CaCl2, 0.25M MgCl2), the ligation or plasmid (up to 15 ul) and water to 100 ul. Then we add 100 ul of competent cells, leave on ice for 20 minutes, and then at 37°C for half an hour, and plate. No heat shock is needed in this protocol.

I routinely get efficiencies of about 10^7 on DH5a (with supercoiled plasmids). You can refreeze and reuse the cells once, but of course the efficiency lowers a bit.

Good luck,

Roberto

=============

Second protocol:

The bacterial transformation mix contains:
10% Polyethylene Glycol(PEG) 3350
PEG 3350 is thought to play several different roles in transformation, though nobody really knows for certain. Since both DNA and cell walls are negatively charged, they reject each other. PEG 3350 is thought to function by shielding the charge of the DNA, thereby making it easier to permeate the cell wall. PEG 3350 is also thought to help transport the DNA into the cell, as well as make the cell membrane itself more porous.

5% Dimethyl Sulfoxide (DMSO)
DMSO is sometimes used to treat ailments in humans. In a transformation it is thought to
permeabilize the cell wall. Also, sometimes DNA folds into complex structures that make it more difficult to pass through the cell wall. DMSO also might help to break these structures down.

25mM Calcium Chloride(CaCl2)
Similarly to PEG 3350, CaCl2 is thought to shield and neutralize the negative charge of DNA, thereby making it more likely to enter into the cell.
1. If you received a plate or stab of E. coli HME63, simply use an inoculating loop to
gently scrape out the bacteria and spread it onto a new LB Agar plate. Let the plate
grow overnight ~12-18 hours, or until you see white-ish bacteria begin to grow. Make
sure you are using the LB agar plate, NOT the LB Strep/Kan agar plate. See the
following link for a walk-through of how to streak out bacteria: https://goo.gl/GR8IOf

 

From:

crispr-bacterial-guide

Pushing the Limits of DNA Assembly

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http://blog.addgene.org/pushing-the-limits-of-dna-assembly

Alternatives to NCBI BLAST during US government shutdowns?

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http://www.insdc.org/

http://www.ebi.ac.uk/

http://blast.ddbj.nig.ac.jp/

https://www.ebi.ac.uk/Tools/sss/ncbiblast/nucleotide.html

http://www.ebi.ac.uk/Tools/sss/ncbiblast/nucleotide.html

https://www.ncbi.nlm.nih.gov/guide/howto/run-blast-local/

 

 

From:

https://biology.stackexchange.com/questions/65225/alternatives-to-ncbi-blast-during-us-government-shutdowns?newsletter=1&nlcode=528019%7c2327

 

Cell surface biotinylation on Lysine residues

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  • EZ-Link sulfo-NHS-SS Biotin (Pierce): cleavable, will bind to all accessible Lysine residues; dissolve in H buffer to 1 mg/ml.
  • H buffer
  • PBS + 1 mM MgCl2 + 0.1 mM CaCl2 with 1% BSA [referred to as PBS(+)-BSA].
  • RIPA lysis buffer (150 mM NaCl, 20 mM Tris-HCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, pH 8.0. With proteinase inhibitors: 10 µg/ml of leupeptine, 10 µg/ml pepstatin, 1 mM PMSF).
  • 5X Laemmli Sample Buffer (diluted from 10x).
  • (optional) MESNA solution for stripping the bound biotin.
H buffer composition, pH 7.6 (It need around 310 ul NaOH 10N to reach Ph 7.6)
final volume (ml): 500
desired [] (mM) MW (g/mol) needed weight (mg)
NaCl 154 58.44 4499.88
Hepes 10 238.3 1191.50
KCl 3 74.56 111.84
MgCl2 1 203.3 101.65
CaCl2 0.2 110.99  (147.02) 5.55 (14.7)
glucose 10 180.16 900.80

 

MESNA (membrane-impermeable reducing agent) solution composition, pH 8.6
final volume (ml): 100
desired [] (mM) MW (g/mol) needed weight (mg)
NaCl 100 58.44 584.4
TrisHCl (maybe means Tris) 50 157.6 (121.14) 788 (605.7)
MgCl2 1 203.3 20.33
CaCl2 0.1 110.99 (147.02) 1.11 (1.47)
MESNA 50 164.2 821

 

10X sample buffer Laemilli (LSB):

12.5% SDS

500 mM DTT (dithiothreitol)

300 mM Tris pH6.8

40mM EDTA

1.75% Glycerol

0.035% BPB (color blue)

Procedure

Seed CFBE-iCFTR WT-3HA cells 100.000/well on filters and keep them >5 days confluent with 250 ng/mL doxycycline on 12 well filter plates.

  • Wash the cells 3 times with ice-cold H buffer.
  • Incubate 15 min with (for 6 cm plate = 0.5 to 1 ml) (1 mg/ml) sulfo-NHS-SS Biotin in H buffer at 0°C. Optional: remove it with suction and repeat it.
  • Rinse twice with ice-cold PBS(+)-1% BSA, and incubate 10 min in PBS(+)- 1% BSA.
  • Optional: perform study of endocytosis.
  • Optional: treat cells with MESNA 3 times 20 min on ice to remove biotin from the cell surface
  • Lyse the cells with(0.5 to 1 ml) RIPA buffer containing protease inhibitor on ice (less than 10 min)
    • Cut out the 4 filters and put into 400 uL RIPA in a pre-cold eppendorf for 5-10 min
    • Vortex throughfully, pipet up and down 5 x and remove the filters.
  • Vortex and centrifuge at 4°C for 10 min at 12000 g (supernatants can be frozen and kept at -80°C).
  • Incubate the supernatant with 20 µl BcMag monomeric avidin magnetic beads (#MMI-102) (wash the beads 3 times with RIPA + protease inh before use) and rotate for 1 h at 4°C.
  • Use the magnetic separator and wash beads by resuspending in 1 ml of lysis buffer. Repeat twice more.
  • Elute the protein with 50 µl + 20 of 5X Laemmli Sample Buffer (diluted from 10x LSB) + 6mM biotin (B4505-1G) and incubate for 10-20 min at RT. Vortex it.
  • Centrifuge the samples at 14000 RPM, RT for 2 min, save the supernatants and perform a 4%-7% SDS-PAGE (when revealing CFTR or other membrane protein, use also an antibody against a cytosolic protein – Hsc70 or other – to check that NHS-SS Biotin has not entered the cells).

 

ImageLab 6.0

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ImageLab 6.0

https://app.box.com/s/z4grf20x1pd4u5df11n8

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