I'm trying to ligate a 60bp DNA insert into PLKO.1 vector (7kb) but I met some problems during ligation.
The vector was double digested with AgeI and EcoRI and purified from gel. And the DNA insert is synthesized by IDT with AgeI and EcoRI overhang.My ligation system is 20ul: 2unit T4 ligase, DNA:insert ratio is 1:3, and I also tried different ratios (1:10).
However, I could not see any colonies on the plate after transformation. My negative control (transformed with double digested vector) also does not have colonies. My positive control (transformed with integral vector) is also working, I could see colonies on the plate.
Could anyone give me some suggestions?
Thanks a lot.
1) Try high-efficiency competent cell
2) Keep your ligation system at 4 degree overnight
3) Before adding your insert, heat a aliquot 30 secs at 95 degree and then cool it slowly at RT to make sure the double strand DNA annealing well.
I agree with Chen's suggestions and heres some more pointers:
FOR LIGATION:
1) when annealing your insert before ligation via slow cooling (as per Chen) you an enhance the procedure by adding 10mM MgCl2 or MgSO4 to stabilize the 60mer duplex. (slow cooling can be done by boiling the primer duplex in a water bath, and turning the bath off, and letting it come to room temperature off its own accord).
2) anneal your insert at 100X concentration so that these added salts do not interfere in Ligation Reaction when an aliquot is added.
3) Add fresh 0.5mM ATP, sometimes older ligation buffers dont work for this reason.
FOR RESTRICTION DIGESTION:
1) make sure your SINGLE DIGESTS are working by gel analysis and make sure you are not overincubating the reactions.
2) EcoRI has star activity, DO NOT incubate the reaction more than 30-45 mins and DO NOT ADD EXCESS enzyme either (use manufacturer recommended amounts for restriction). BOTH of these are deleterious for subsequent ligations as it destroys sticky ends.
3) Gel purify your double digested DNA (make sure you observe using long wavelength UV, short wave will crosslink the DNA and make it unligatable) and Dissolve your final elute in sterile Nuclease free water. \
FOR TRANSFORMATION:
1) your positive control should be done with 1ng of intact plasmid - and your transformation efficiency (no of transformants/ug of DNA used) should be more than (10) raised to 5 or 6 to see ligatable transormants.
2) if you are using CaCl2 transformation and getting low efficiency, move to electroporation.
When purifying anything from gelelektrophoresis make sure that you remove any traces of agarose since it inhibites ligation. At least that was the problem with my transformations. To ensure that agarose was completely removed i tripled the washing steps.
Hope it helps.
http://www.addgene.org/tools/protocols/plko/#A
There is either a problem of ligation or transformation. To check ligation, you can do a PCR on an aliquot of your ligation mix using primers adjacent to the EcoR1 and Age1 cloning sites. Unfortunately the sequencing primers in PLKO.1 are quite far away from the site you are cloning into, so you will have to design your own primers. This will confirm if you have ligation of the right sized products. If the ligation is OK then the issue is transformation. You may need to extend the growth period without Ab to allow "recovery" before applying selection and plating on plates. (alternatively, keep back a small portion of your transformation and grow in culture media o/n with Ab and then plate out).
Is your synthesized DNA in a vector or supplied as dsDNA? If it is the latter and it has overhangs, it could be that the overhangs are being degraded by DNAses. When inserting such short sequences into vectors, I have always ordered single stranded oligos, annealed them and then inserted into a digested ( and gel extracted) vector through ligation. Just make sure you design each oligo so it will have the required overhangs when annealed. Hope it helps.
If there is problem with Insert, your vector control should give at least few colonies because it is double digested.
Make sure that the gel purification was done properly , spin twice and carefully remove the supernatant.
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