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

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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

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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

Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

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http://blog.addgene.org/tips-for-a-1st-time-crispr-user

Cancer and the Immune System: Deciphering the Relationship

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http://blog.addgene.org/cancer-and-the-immune-system-deciphering-the-relationship

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