I have done transformation of pIndie-C1 DNA (using 100 uL ; 0.6 ug/uL) in Top 10 competent cells using heat shock method. 400 uL was transferred to 250 uL LB broth and 100 uL was plated in LB agar. Then I have incubated for 15 hours in shaker at 30*C and 200 rpm. The colonies appears after long time. 20 hours and they are very less. What is the reason for very slow growth and what can be done for good yield of cell growth with plasmid.
At 30*C, with a relatively large plasmid (as is your case) and TOP10 cells, 20 hours until colonies become visible is not unusual. The low transformation frequency probably has to do with a combination of large plasmid size (transf. freq. decreases linearly with plasmid size, according to the original 1983 Hanahan paper) and low frequency competent cells. Next time include a control transformation with 1 ng of pUC19; it should yield at least 10e7 cfu/ug DNA.
You might be able to increase yields by amplification with chloramphenicol; see e.g. http://www.bio.net/bionet/mm/methods/1992-August/002283.html or find a copy of Sambrook's manual.
Prepare competent cell as in the attached protocol, except for aliquots size: make them of 200 uL each. You will use them for several trasformation.
You can scale-down all the volumes starting from point 4 (e.g. 200 mL cultures instead of 500 mL) if you don't need too much aliquots of competent cells, also allowing you to centrifuce cells in sterile 50 mL Falcon.
- Thaw out one aliquot at room temperature
- Add 1-2 ul of prepped plasmid (should not exceed 100-150 ng of DNA)
- Hold on ice 30 min
- Heat shock 50-55'' at 42° C (not less than 42'', not more than 60'')
- Add 800 ul SOC (LB is also suitable)
- Shake at 37° C in 2 ml centrifuge tubes rotated (like this
I got the reason for my mistake. I used the improper DNA concentration (too much) at that time. And I used 30°C instead of 37°C as at 37°C there is more chance of efflux mechanism of E. coli competent cells (TOP10).
Please suggest me trasfection criteria with pINDIEC-1 DNA to Hela, HEK-293 cells.
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