Dear biology scientists,
Hello, I should do transduct lentivirus in the 293FT (HEK293) cells soon.
However, I have failed tranformation because the DH5a colonies were too low.
Let me tell my experimental procedure as below.
1. Making 10 mL of LB agar plate without any antibiotics
2. Spreading 10 mg/mL of Ampicillin in the 10 mL of LB agar plate
3. Melting the Escherichia coli DH5a in the ice
4. Aliquoting the DH5a by 30 uL per each sample
5. Inserting a vertor sample in the DH5a by doing spiral pipetting
6. 30 min incubation in the ice
7. 42 degree Celcius for heat-shock
8. 2 min incubation in the ice
9. Putting 1 mL of LB broth without any antibiotics into the heat-shocked DH5a
10. 37 degree Celcius incubation in a shaker for 45 min
11. 13000 rpm, 2 min, room temperature centrifuge
12. Spreading 100 uL of supernatant in the LB agar plate with 100 ug/mL Ampicillin
13. 37 degree Celcius incubation in an incubation
I have no idea why my colonies were rarely shown.
Another person did my procedure, and she got many pMD2G colonies and psPAX2 colonies.
What is my problem? Please help me.
When you have low transformation the problem usually is low amount of plasmid DNA or (most probable) cells that are not very competent. You can do a control transformation with another plasmid that you know is good quantity to test for the competent cells. You can run your plasmid on a gel to see whether there is ample plasmid in your preparation.
I think you don't have enough plasmid. I have had this issue before. What I did was follow the same protocol using the same cell line and did a control with a plasmid that worked in the past. I realized my plasmid wasn't enough. So to confirm if you have enough plasmid or if your DH5a is competent, do a control with a plasmid that you've transformed in the past (and it worked) which will serve as your control and follow the same protocol you're working with. This will tell you if your current plasmid is enough or if your current cells are competent.
At step 12, try to remove about 500ul supernatant, resuspend the sedement and then take 100ul. It usually works with me
Any one plz guide I have E. coli DH5 alpha competent cells and i have purchased it from invitrogen thermo fisher scientific. Can i sub-culture them and store for future use if i run out of stock. Buying commerical cells again and again is very expensive
Yes you can streak out a small amount and save it. But you will need to make them competent yourself and it can be difficult to get the same level of efficiency of competence as the commercial ones.
Sure you can.
Here is the protocol I use.
Preparation of competent E. Coli cultures
Important: Do not use any antibiotics for DH5a.
Inoculate an LB culture with DH5α cells (directly from the frozen stock without thawing) and grow overnight at 37 °C.
1. Inoculate 5-6 colonies in 100ml SOB medium in 1L flask, grow to OD600~ 0.3-0.6 at 18°C, (250 rpm). Do not exceed 0.6.
2. Incubate on ice for 10 minutes
3. Centrifugate (2,500g 10'min at +4°C).
4. Resuspend the sediment in 32ml of cold TB.
5. Incubate on ice for 10 minutes
6. Centrifugate, 2,500g 10 min at +4 °C.
7. Resuspend the sediment in 8ml of cold TB.
8. Add 560ul DMSO (carefully, slowly, I recommend add it on the tube wall while slowly rotating the tube).
9. Incubate on ice for 10 minutes
10. Dispense the suspension into sterile microcentrifuge tubes (100 ul is enough ph).
11. Freeze in liquid nitrogen
12. Store at -80°C
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