Hello there guys,
I am cloning into a lentiviral vector but unfortunate I am not getting any colonies.
I restrict the CD55 (50 nanograms of DNA) gene using both AgeI and XhoI and I restrict the vector (1 Microgra) using SalI and Agel both reaction carried in NEBuffer 2 And then incubate for an hour at 37
And then I do the electrophoresis and extract the DNA from the gel.
I treat the vector using Antarctic Phosphatase for 30 min (20 incubation at 37+ 10 deactivation at 65)
I then use the T4 ligase with ration (1 vector :3 insert) over night at 4 C' and then the next day in the morning I transform the cells and plate then in selection plate at 30 C' over night
what am I missing ?
Hi Ahmed,
Firstly - are you using NEB T4 ligase with its cognate buffer? I am hoping so given that you are using NEB enzymes for the other processes described. Please make sure if so that the buffer is very fresh (smells strongly of DTT and remains in date) because the ATP content is very sensitive to improper freeze-thaw discipline, and this is essential for successful ligation. Also - never vortex ligase buffer. Flick it while it is thawing, but anything else will cause the ATP to deteriorate quickly.
Secondly, as suggested by Dr. Ding above, you are being a little conservative with preparation of your insert and plasmid. Because AgeI and SalI do not have compatible ends, there is no good reason to use phosphatase (whether Antarctic or CIP) on the vector - it is just another complication. If you are Phosphatase treating because your vector is religating without insert - you need to make sure that the digestion is complete and solve this problem before proceeding to the ligation step.
My gut instinct is that the major problem is improper or incomplete digestions because of incorrect choice of buffer / reaction conditions.
For instance - unless you have HF-engineered versions of either AgeI or SalI, digesting in buffer 2.1 is probably going to be extremely inefficient as neither enzyme is optimal in this buffer. Moreover, NEB's own online double-digestion tool suggests that these two non-HF enzymes should be used sequentially rather than together.
Secondly, even the insert digestion seems to be compromised by the buffer choices - NEB's own tool suggests that AgeI and XhoI should be digested in 1.1 or Cutsmart, although 2.1 is a similar score (but they probably have a reason for their suggestion).
Secondly, the amounts of DNA. I would digest considerably more insert - if this is a typical miniprep, I would digest 16 microlitres of DNA in a 20ul digestion for 1 hour, then resolve by agarose gel electophoresis to separate the insert band.
Vector - assuming you have resolved the enzyme / buffer question and it digests to completion, try 5ul in a 20ul digestion. If this is too strong when you run the gel, either dilute the prep or reduce the DNA input further until you get a clean band. If in doubt, perform single digests in parallel to show that both enzymes are digesting separately in those buffer conditions
When you are satisfied that insert and plasmid are both correctly digested, then gel purify both.
Ligations - if you have good NEB T4 ligase with fresh buffer, and good DNA for both components, ligation should take no longer than 15 minutes at 20-25 centigrade. I haven't done an overnight ligation since 1996.
Set up various insert to vector ratios, as well as empty vector control, vector without ligase control etc in 20ul volumes (when ligations are working I use 10ul to reduce use of ligase enzyme). After 15-30 minutes, chill the reactions, and transform to freshly-thawed chemically-competent E.coli using standard protocols, and plate to appropriate antibiotic-LB agar after 2-3 hours recovery in LB broth. Hopefully you should get a good ratio of colonies in ligations to ligation controls.
In short : sort out the digestions and follow best practise when it comes to basic Molecular Biology. NEB have tools on their website and probably know how their enzymes work best, so inventing novel protocols with inappropriate buffers is probably not the best way to succeed, however much you should be lauded for enthusiasm.
Please feel free to discuss this here further - and don't take my comments as being critical : we all have been in this position at some point in our careers, and it is through things not working that we learn the most. Good luck!
Hui Ding
Thanks alot i think after the gel extraction i get somewhere between 20-30 Nanogramms
Robin Dickinson Thanks alot mate, I am super grateful,
I will try to edit the protocol i am working with and if anything comes up i will let you know,
Again i am greatly thankful
You're welcome. Happy to discuss further when you have progress or if you have any protocol questions while you're editing.
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