He Tube Mating with MSCV recipient

Chemicals needed:

Glucose  

L-(+)-Arabinose   Sigma Cat # A3256

L-Rhamnose  Sigma cat # R3875

Cb (carbenicillin)  Sigma cat # 1389

DL-p-Chlorophenylalanine  Sigma cat # C6506

Media Recipes:

YEG recombination plates:

1 liter Milli Q water

5 g yeast extract

10 g NaCl (5 g for any Zeocin recombinants)

2 g DL-p-Chlorophenylalanine powder (will not dissolve before autoclaving)

20 g agar

AUTOCLAVE

After media has cooled to 55℃ add the following and stir gently:

8 ml 50% Glycerol (sterile)

10 ml 20% L-arabinose (filter sterile)

1 ml Carbenicillin at 100mg/ml (for pMSCV selection)

AND antibiotic to select for your insert

POUR

YEGlu:

1 liter MilliQ water

5 g yeast extract

10 g NaCl

autoclave and cool

add 8 ml of 50% Glucose (filter sterile)

Strain and vector information:

Recipient pMSCV:  PheS gene flanked with 2-Isce-I site and homologous sites for recombination, 10.6 kb. Carbenicillin (amp) resistent.

Recipient Strain BW28705I/pML300:  pML300 plasmid carries the recombinase driven, temperature sensitive rhamnose inducible PrhaB-gbexo promoter. This promoter is inhibited by glucose. This strain also contains an arabinose inducible promoter paraBAD controlling expression of the I-sceI gene. Induction of I-SceI gene allows for the cleavage of library cassette out of donor plasmid. Presence of glucose represses the paraBAD promotor.  pML300 is Spectinomycin (50).

Donor bacterial strain:  BW F’,  the F’ is Tet (1).

Donor vector : Most likely pSHAGMAGIC: insert cassette is between the H1H2 recombination regions which lie just inside two I-SceI cut sites (Sce1-H1-Cassette-H2-Sce1). Vector is Kanamycin (20), hairpin library insert is Chloramphenicol (50).

Tube Mating Protocol

Preparation:

1. Streak fresh donor plasmid on LB + proper antibiotic +IPTG/XGAL plate (since the donor F’ strain contains lac +, it will be blue on the XGAL plate

2. Streak recipient plasmid (pMSCV) on LB+Spec50+CB100+0.2% glucose OR YEGlu+spec50+cb100) Grow at 30℃ overnight

Protocol

1. Begin 5 ml overnight cultures of recipient, donor and control donor.  Grow ALL at 30℃ shake

Donor and donor control: LB+ Kan20+Chlor50 (your insert may differ)

Recipient: YEGlu+spec50+Carbenicillin100 (glucose represses promotors you don’t want turned on for replication in an o/n culture

2. The next day you need to wash the glucose out of the recipient. Pellet on culture by spinning 4K for 5 min. Resuspend the pellet in 10 ml LB and spin 4k 5 min. Repeat this step for a second wash. Resuspend pMSCV in 5 ml LB.

3. Set up Dilutions:  Set up dilutions as shown below, in 5 ml LB+0.2% rhamnose. Use 50 ml Falcons for this as they have good aeration and you get much better growth. Place these dilutions at 30℃ shaking for 2 hours.

pMSCV:  25 μl ? 5ml 

  50 μl ? 5ml

  100 μl ?5 ml

Donors:  25 μl ? 5ml 

  50 μl ? 5ml

  100 μl ?5 ml

  200 μl ?5 ml

4. After the 2 hour shaking at 30, place 2 mls of each culture into a cuvette and read OD600. The optimal OD range is .080 to .200, but when given a choice, use the dilution with the OD closest to 0.1 (this is true for recipient AND donor cultures. You want an actively growing culture for optimal mating)

5. To set up tube mating, you want a 1:1 ratio of recipient:donor. Determine which dilution of the MSCV dilutions you will use, then multiply by 500. Divide this by the OD600 of your Donor, and you will get the amount of donor in μl to add to 500 μl of MSCV:           (OD600MSCV)(500 ul) ? OD600 of donor culture = μl of donor culture to use

6. Mix 500 μl MSCV culture with the calculated amount of donor culture above in a 50 ml Flacon.  Determine final volume of mix and add 20% arabinose to a final concentration of 0.2%. (1 ml mate culture gets 10 μl 20% arabinose)

7. Place your mating cultures at 37℃ NOT SHAKING for 2 hours. This is the actual mating step, induced by arabinose

8. Move to 37℃ shaker for another 2 hours. This grows all forms of mated recombinants and unmated bugs

9. Make 10-1, 10-2 and 10-3 dilutions for each mating, and plate 100 μl of each dilution onto PREWARMED  plates of YEG recombinant plates.  PLACE AT 41oC overnight.

NOTES: Temp is crucial. Overshooting temp is detrimental, its better to have temp 1-2 degrees lower than 42. However, you may incur higher background of pMSCV if the temperature is too low.

Plates are also crucial as positive and negative selection happen at the plating stage. Use fresh plates and do not let them dry out during prewarming. Dried out plates will cause you to lose colonies and wet plates will make colonies run together.

To calculate efficiency of the mating, count the colonies on the plate.

# colonies X dilution X 10 =   # colonies/ml

a high efficiency is  107 colonies/ml