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Team:Nevada/Transformations
From 2010.igem.org
NT Cell Transformations
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Protocol
CARE OF A NT CULTURE (adapted from a letter by Dr. Michael Sullivan) To readapt a culture on plates, simply transfer some of the cells back into liquid media. We usually pipette the cell suspension up and down to break up any clumps. It may be best to start out with a smaller culture volume when you first go back to liquid; BY-2 seems to be a bit happier if it isn't seeded to thinly. Allow the culture to grow until it is the consistency of thin applesauce. At this point, you should be able to go to a 50 ml culture and start subculturing as described below. In our experience, wild-type BY-2 readapts quickly to liquid to give a smooth culture; transformed lines tend to be more variable, with some producing smooth cultures, some producing clumpy ones, and some going back and forth between these two states. We've found that clumpy cultures do not interfere with our half-live measurements, although manipulating them can be a bit more difficult.
We grow our liquid cultures in 50 ml of media in 250 ml baffle flasks at 28 degrees C with gentle shaking (150 rpm). Using a baffle flask does not seem to be a requirement, many people grow these cells in regular flasks with no problem. We subculture them once a week by transferring 5% of the culture to fresh media. We generally maintain two flasks (in two separate shakers) of two separate subcultures (one subcultured Monday, one on Friday) in case one of the cultures crashes. Also, you can maintain the culture on a plate. Note that, for liquid cultures of transformed lines, we usually use a smaller culture volume, e.g. 10 ml, for convenience.
NT KC MEDIA - LIQUID OR PLATES
NT LIQUID:
(amounts are for 1L of media):
750 ml H2O
4.3 g MS salts (add slowly to liquid)
30 g Sucrose
50 ml 20X MES pH 5.7
10 ml B1 -inositol
3 ml Miller's I
10 ml 2,4-D, 10-4 M
pH to 5.7 with 0.1 N KOH
Bring volume to 1000ml
Autoclave
SOLID MEDIA: For plates only: Add to flasks 7 g/ L Phytagar before autoclaving Add kanamycin (100 mg/ml) Add carbenicillin (250 mg/ml)
MEDIA COMPONENTS: Miller's I: 60 g KH2 PO4 per liter 20 X MES: 10 g MES per liter, pH to 5.7 with 1N KOH B1 - Inositol: 0.1 g Thiamine, 10.0 g myo-inositol, H2O to final volume of 1 liter
TRANSFORMATION PROTOCOL:
NT Cell Transformation with Agrobactrium
Day 1:
1. Grow up 1 ml of the Agrobacterium overnight in LB + all selective drugs. This culture may be started from frozen glycerol cultures if necessary.
Day 2: 2. NT cells are used 3 days after splitting the NT cell culture. 4 ml of NT cells are required for each transformation with an additional 4 ml for the control culture which receives no bacteria.
3. 1 ul Acetosyringone (20 mM in ethanol) is added per ml of NT cells. Typically treat the whole 50 ml culture at this point and discard any that is left over when I'm finished.
4. Using a 10 ml pipette, the NT cells are pipetted in and out about 20 times. This helps to induce small lesions in the cells and increases the efficiency of the transformation.
5. 75 ul of bacteria (dense growth) or 100 ul (moderate growth) are added to a petri dish containing 4 ml of NT cells (from step 4) and mixed thoroughly. REMEMBER TO INCLUDE A CONTROL HAVING NO BACTERIA.
6. Wrap plates with parafilm and incubate for 3 days at 28 degrees C.
Day 5: 7. To each plate add 10 ml of NT liquid medium containing 500 (u)g/ml carbenicillin (NTC).
8. Collect cells off of the plates into 50 ml conical tubes and fill with NTC.
9. Centrifuge at 1K for 4 minutes at room temperature in a swinging bucket rotor.
10. Aspirate off liquid using pipet capped with a sterile blue 1 ml top. Change tip for each sample.
11. Resuspend in 50 ml NTC and repeat spin.
12. Repeat step 10 and 11 - 1 or 2 times. Strains that are fairly sensitive to carb require fewer washes.
13. Resuspend cells in approximately 5 ml NTC and plate 2.0 ml on 2 NTKC (kanamycin 100 ug/ml carbenicillin 500 ul/ml) plates. When there is no longer lots of fluid on the plates (leave the plates open in the hood a few minutes of necessary), wrap them in parafilm.
14. Incubate at 28 degrees C. Transformants should be large enough to transfer to fresh NTKC plates in 3-4 weeks.
Supplies for each transformation (Remember the controls):
Day 1 Supplies: LB + appropriate drugs Agrobacterium containing plasmid for transformation
Day 2 Supplies: 1 ml Agrobacterium overnight culture 4 ml BY-2 cells - 3 days post subculture 4 ul acetosyringone (20 mM) found in the (-20) refrigerator freezer pipets and pipetman 1 petri plate
Day 5 Supplies: 200 ml NTC liquid 50 ml conical tube swinging bucket centrifuge at room temp aspiration setup with 5 ml pipet capped with 1 ml blue tip 2 NTKC plates pipetmen and tips
Left: NT Cell Culture after about 5 days. Right: NT Cell Transformation with RD29A (Cold, Drought, Salt Induciblbe Promoter.
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