Hello everyone, I have one doubt regarding cloning ?
I am using pET30a (+) vector for cloning (5422bp) and Insert size 804bp. I tried all the ligation reaction ratio lilke 1:3, 1:5, 1;7 (100ng vector and 300ng Insert) but some time I got positive colonies but the problems is that whenever i am going to isolate the plasmid, I donot found the plasmid in the positve colonies
Please give me some suggestion
what should I do for it
The step you are missing in your calculation is adjusting the length of DNA (insert and vector) to get molar ratios:-
You insert DNA amount must be adjusted based on the length of the DNA (insert) and vector to get a molar ratio.
For example : for 1:3 ratio
Formula is : Insert amount (ng) = Vector amount x Insert size / Vector size x (Molar ratio)
Insert amount (ng) = 100ng x 0.804 (kb) / 5.422 (Kb) x (3) =44.48ng
Thus for 100ng of vector , you require 44.28ng of your insert (for 1:3 ratio)
You say that you get positive colonies, on what basis do you say that? It may be that you are not getting any positive colonies at all.
How are you checking the colonies for presence of the plasmid? I assume that you are streaking and also making a liquid overnight culture? Then plasmid prep, then PCR. Sometimes, you have to linearize the plasmid to get efficient PCR. Pick a rare cutter that you know will NOT cut in the insert.
Double-check that the antibiotic you are using for the overnight growth is the correct concentration. It's really easy to "drop" a plasmid if you remove the antibiotic selection.
Good luck!
Colony PCR is notoriously unreliable. It's easy to get false positives (plasmid on media) and false negatives (unable to amplify plasmid, even when present).
I'd recommend the following:
1. Choose colonies. Number them & streak to make a reference plate, also set up overnight liquid cultures with selection.
2. Use the liquid cultures to extract plasmid. "boiling lysis" or a kit both work.
3a (optional, but ideal) Linearize plasmid using rare cutting that does NOT cut in the insert.
3. Set up PCR using 1 primer in plasmid + 1 in insert OR both primers in plasmid that flank the insert. Include a known "empty vector".
4. Check for size of amplicon on agarose gel.
5. If needed, repeat PCR to amplify entire insert for sequencing PCR.
Keep (total volume 20 mL) ligation reaction 30 min at 25 C and then add 0.5 uL of 100 mM ATP in the ligation reaction, mix by pipetting, keep 1 h at 25 C then keep it in refrigerator (4-8 C) over night, then transform, do not tap or mix DNA in comp cells, just add them in the center of the cells (add 8 uL of Ligation mixture to 40 uL of comp cells) follow everything same way then last step incubate 45 min minimum on shaking 37 C, before plating. Let me know if that works! If it doesn't work then change the primers length a little bit and order new one, might be bad lot or synthesis error. If you can afford and above doesn't work then same steps do with commercially purchased comp cells. I got these one and worked. https://www.thermofisher.com/order/catalog/product/18265017 Subcloning Efficiency™ DH5α Competent Cells Catalog number: 18265017 then I made my own from growing from them and was working.
Ligation Mixture: (Total DNA; Vector and Insert must be no less than 50 ng). Suppose you got gel extracted vector (5ng/ul) and insert (3 ng/ul) then make sure the total DNA should be no less than 50ng in ligation reaction mixture by whatever ratio you use, I generally use 1:2 and it works. Always gel extract your vector and insert in EB buffer (It's mostly Tris buffer from vendor kit) keep EB buffer on 50 C for 10 min before gel extraction and add only warm EB buffer (take it from 50 C right before adding to elute) 5 ul first elution with 2 min spin on 13K RPM and second elution same way. upto 20 µL Water n µL Vector DNA n µL insert DNA 2 µL T4 DNA ligase buffer 0.5 µL 100 mM ATP 0.5 µL T4 DNA ligase Mix Well 1. Gently mix the reaction by pipetting up and down and microfuge briefly. 2. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 60 minutes. 3. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours *(alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation)*. Transform into JM109/DH5a (DH5a was used) competent cells. Control No Insert. Just add 8 µL of DNA mixture to 40 µL of competent cells just add all in the center of the cells, do not mix or tap. 1: Competent cells thaw 20 min on ice from -80 °C stock to thaw. 2: Add Ligation mix 8 ul. 3: Incubate for 30 min on ice. 4: Heat Shock 42°C for 60 sec 5: Keep back on ice for 2 min. 6: Add 250 μL LB media 7: On Shaker 250 RPM incubate at 37°C for 60 min. 8: Plate of respective Antibiotic (Kan/Amp) all 250 μL.
If still doesn't work just add 1 μL of 100 mM ATP, if that too doesn't work try more higher concentration, you can use 0.5 to 2 μL of 100 mM ATP in one go and check if you are getting ligation work or not.
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