Dear professors and colleagues
Situation:
I am currently preparing a recombinant DNA consisting of a 1000bp insert and a 6000bp vector.
- Insert: The 1000bp insert is prepared by amplification using PCR and then double-digested to create sticky ends.
- Vector: The 7500bp vector has been double digested using the same restriction enzymes and then extracted and purified 6000bp part from 0.6% agarose gel.
Trouble:
1- After extraction and purification the final concentration is 12-20ng/ul and the desired concentration to perform successful ligation is 100ng/ul or more. What is your advice to get a high concentration from the gel?
2- After proceeding to ligation using different protocols and companies,
insert to vector ratio is 3:1 using 270ng/ul of insert and 12ng/ul of vector; the end result in gel electrophoreses is way longer than the desired length by approximately 10000bp.
Do you have any suggestions?
I will appreciate your opinion and advice....
We purify DNA at several steps i.e., after PCR and after digestion, You can purify PCR product and vector at every step from solution instead of purification from gel if there is no non-specific DNA. If there is some unwanted DNA you can purify DNA from the gel only for that particular step. It will help you to get better concentration. Which ligase are you using? If you are using T4 DNA ligase, I will suggest you heat the ligation reaction (without T4 DNA ligase) mixture at 65 degrees Celsius for 3-5 minutes, immediately cool down the reaction mixture on ice (for ~5 minutes), and then add T4 DNA ligase. If you are using handmade competent cells prepared by the Ca-Mg method, I will suggest you prepare competent cells by the Inoue method (ultra-competent cells) else you can prepare them by electroporation if you have that facility. I hope it will help you to get results. Wish you the best!
Run your PCR for more cycles and maybe add more NTPs. Also you can run multiple replicates of the PCR/DNA purification and use the same batch of elutant for all the columns to get a higher concentration. Put the elutant through the columns twice with a 1 min incubation for about 50% higher yields. The large band is indicating you are forming concatemers so you may want to perform your ligation in a larger volume.
Pls treat your vector with phosphatase to avoid ligation of two vector molecules by cohesive ends.
Some agarose gels give poor recovery of fragments. 12ng is OK for ligation, this is not a truoble, just fewer clones after transformation
dear Ahmed Elsherbini to point 1: your concentration is fine. You don't need more than that. In general people use 100 ng of vector for a ligation IN TOTAL (that is about 8 ul of your purified fragment of 12ng/ul, which you should have; but you can also use consideraby less, it depends how efficient your cells are for transformation). Point 2; the 3:1 ratio vector to insert is NOT about ng but about molar ratio. that means 3 molecules insert vs 1 molecule vector. Your vector is 6kb, your insert 1 kb, so for 100 ng vector you need about 50 ng insert. So in summary: probably u use way too much insert. I don't know what u mean about the end product in electrophoresis? After transformation and miniprep? Maybe you get vector dimer, it can happen. As Anatoliy said, try dephosphorylating the backbone vector prior to purification. If after fixing the ratio, problem remains, make sure the sites in your PCR fragment are properly cut (enough base pairs at end next to restriction site etc).
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