Team:TUDelft/Protocols

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Contents

Prepering chemically competant cells - TSS Buffer

Materials

Glassware & Equipment

  • Falcon tubes
  • 500μl Eppendorf tubes, on ice
  • 200ml conical flask
  • 200μl pipetman or repeating pipettor
  • 5ml pipette

Preparation

  1. Grow a 5ml overnight culture of cells in LB media. In the morning, dilute this culture back into 25-50ml of fresh LB media in a 200ml conical flask. You should aim to dilute the overnight culture by at least 1/100.
  2. Grow the diluted culture to an OD600 of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD600 0.2)
  3. Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is X ml, you will need X tubes. At this point you should also make sure that your TSS is being chilled (it should be stored at 4°C but if you have just made it fresh then put it in an ice bath).
  4. Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.

All subsequent steps should be carried out at <math>4^o</math>C and the cells should be kept on ice wherever possible

  1. Centrifuge for 10 minutes at 3000 rpm and <math>4^o</math>C.
  2. Remove supernatant. The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful. Pipette out any remaining media.
  3. Resuspend in chilled TSS buffer. The volume of TSS to use is 10% of the culture volume that you spun down. You may need to vortex gently to fully resuspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall.
  4. Add 100 μl aliquots to your chilled eppendorfs and store at <math>-80^o</math>C.
    • The original paper chung suggests freezing the cells immediately using a dry ice bath. I (BC) have used liquid nitrogen quite successfully instead of dry ice. Simply placing the cells at <math>-80^o</math>C also seems to work well (Jkm)
  5. It is a good idea to run a positive control on the cells.
    • The Endy Lab is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out here.

Related topics & References

Based on a protocol from Kathleen McGinness, annotated by Josh Michener & Barry Canton. Original protocol published by Chung et al.chung chung2

<biblio>

  1. chung pmid=2648393
  2. chung2 pmid=8510550

</biblio>

Prepering chemically competant cells - TMF Buffer

Materials

Glassware & Equipment

  • Falcon tubes
  • 500μl Eppendorf tubes, on ice
  • 200ml conical flask
  • 200μl pipetman or repeating pipettor
  • 5ml pipette

Preparation

  1. Grow a 5ml overnight culture of cells in LB media. In the morning, dilute this culture back into 40ml of fresh LB media with 0.8 ml of Mg-mix (0.5M Magnesium chloride + 0.5M Magnesium sulfate) in a 100ml conical flask. You should aim to dilute the overnight culture by at least 1/100.
  2. Grow the diluted culture to an OD600 of 0.5 - 0.8.
  3. Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is X ml, you will need X tubes. At this point you should also make sure that your TMF is being chilled (it should be stored at 4°C but if you have just made it fresh then put it in an ice bath).
  4. Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.

All subsequent steps should be carried out at 4°C and the cells should be kept on ice wherever possible

  1. Centrifuge for 15 minutes at 4000 rpm and 4°C.
  2. Remove supernatant. The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful. Pipette out any remaining media.
  3. Resuspend in 4ml chilled TMF buffer and add 1 ml of 40% glycerol. You may need to vortex gently to fully resuspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall.
  4. Add 100 μl aliquots to your chilled eppendorfs.
  5. Flash freeze the eppendorfs containing the cells with liquid nitrogen.
  6. Store the cells at -80°C.
  7. It is a good idea to run a positive control on the cells.

Bacterial transformation

Introduction

Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. Bacterial cells into which foreign DNA can be transformed are called competent. Some bacteria are naturally competent (e.g B. subtilis), whereas others such as E. coli are not naturally competent. Non-competent cells can be made competent and then transformed via one of two main approaches; chemical transformation and electroporation.

There are advantages and disadvantages to both transformation methods. In general, chemical transformation is less prone to error and faster however electroporation produces a higher transformation efficiency (fraction of transformed cells that actually uptake the foreign DNA). See Molecular Cloning for a fuller discussion of both approaches.

Protocols

OpenWetWare already has a number of protocols relating to bacterial transformation but more are always welcome.

If you use a variant on one of these protocols please feel free to add a link to your protocol from one of these pages so other users can find a protocol that works for them. Additionally, if anyone uses the Innoe or Hanahan high-efficiency protocols, then please add protocols here.

Chemical transformation

If you plan on doing a chemical transformation, then you should see these pages -

Chemical transformation buffer comparison

Someone should check out the claims of Nishimura90. tk 08:58, 25 September 2007 (EDT)

Rubidium chloride transformation protocol here


Someone should check the claims of 1e10 chemical competence using 10% ethanol and calcium chloride protocols here.

Electroporation

If you plan on using electroporation, then see these pages -

References

<biblio>

  1. MolecularCloning isbn=0-87969-577-3
  2. Hanahan91 pmid=1943786
  3. Nishimura90 pmid=2235524
  4. Hanahan89 US Patent 4,851,348 Media:pat4851348.pdf
  5. Jessee90 US Patent 4,981,797 Media:pat4981797.pdf
  6. Donahue01 US Patent 6,247,369 Media:pat6274369.pdf
  7. Greenr04 US Patent 6,706,525 Media:pat6706525.pdf
  8. Donahue04 US Patent 6,709,854 Media:Pat6709854.pdf
  9. Bloom04 US Patent 6,709,852 Media:pat6709852.pdf
  10. Bloom05 US Patent 6,855,494 Media:pat6855494.pdf
  11. Jessee05 US Patent 6,960,464 Media:pat6960464.pdf
  12. Cohen-PNAS-1972 pmid=4559594

// This appears to be one of the earliest papers on transformation of Escherichia coli. If you find an earlier paper, please include it here.

  1. Sharma07 Sharma AD, Singh J, Gill PK; Ethanol mediated enhancement of bacterial transformation, Electronic Journal of Biotechnology (2007), 10(1), DOI: 10.2225/vol10-issue1-fulltext-10, here.

</biblio>

Medium preparation

TBE preparation

Purpose

  • TBE is used for electrophoresis of nucleic acids in sequencing PAA gels or agarose gels.

Recipe

10x TBE (1 liter):

  1. Dissolve 108 g Tris and 55 g Boric acid in 900 ml distilled water.
  2. Add 40 ml 0.5 M Na2EDTA (pH 8.0) (alternatively use 9.3 g Na2EDTA)
  3. Adjust volume to 1 Liter.
  4. Store at room temperature.

Note: 10x TBE may take some time to dissolve, even with fast stirring

LB Medium

Summary

Luria-Bertani Medium (aka L-Broth or LB Medium). (Bertani says LB really stands for lysogeny broth.) LB is a standard growth medium for a variety of bacteria and conditions.

Ingredients

  1. 10 g Bacto-tryptone
  2. 5 g yeast extract
  3. 10 g NaCl

See also: Silver: LB Liquid

Note: There are two formulations of LB, Miller and Lennox, that differ in the amount of NaCl. Lennox has less salt, only 5 g/L. The Qiagen miniprep kit recommends LB with 10 g NaCl for highest plasmid yields.

Protocol

  1. Mix dry ingredients and add distilled water up to 1 Liter
  2. Pour into 2 L flask (or greater)
  3. Autoclave (liquid cycle)
    • 250°F, 22psi, 30 minutes

Notes: We do not pH medium when we make it on the fly. However, if it is really important, pH the medium to 7.0 with 5M NaOH (~200µL). We usually obtain this from the kitchen.

Source

Adapted From:

J. Sambrook, D.W. Russell, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, ed. 3, 2001) pg. A2.2

Lock/key synthesis

Protocol for Lock/key synthesis

Conjugation Protocol

See Conjugation Protocol.