Hi, I have been using Thermo Scientifics Maxima H Minus cDNA double stranded synthesis kit to generate dscDNA. I carried out 2 rxns with oDT and RH primers (10 pmol) and got ~10 kb band for oDT and a smear, and a relatively long smear for RH on agarose 1%. Both PCR products were purified using Mashery-Nagel kit. I tried twice to clone them with no success. Other products which i cloned along with ds CDNA for control provided colonies.
Does anybody here has an experience using this kit / anything similar ? Any advise would be greatly appreciated.
Sofia
That looks pretty similar to the image from the kit protocol: http://www.thermoscientificbio.com/uploadedImages/Products/Molecular_Cloning/Kits/k2561_1.jpg
Which suggests your synthesis is ok.
You could of course check it by trying to PCR a couple of genes you know should be expressed: if you can pick those up then you're good to go.
This thus means you're probably having problems with your ligation. Have you specced your purified dscDNA to confirm it's clean? (260/230 ratio is important here: lots of PCR cleanup kits use guanidium -I think MN's kit does-, which is ludicrously inhibitory to downstream reactions). Also, make sure your vector is compatible with blunt-ended ligation: I'm assuming you phosphatase treat your vector to prevent re-ligation, but this might mean you need to add back phosphates to your dscDNA fragments (T4 polynucleotide kinase, for instance).
Could you provide more detail? This is a little unclear.
Are you making dscDNA, and then using a small amount of this in a PCR with specific primers designed to your gene of interest, then running that on a gel, then cloning the band from that?
Or are you just running your sdcDNA on a gel directly?
Your description of the banding patterns sounds a bit like the latter: there are limits to reverse transcriptase processivity, so oligodT (working exclusively from 3' to 5') will often produce a range of cDNA sizes with a fairly consistent upper limit (so faint upper band + smear), while random priming can begin anywhere, so will simply produce a wide range of cDNA sizes (smear).
EDIT: and if it's the former, what cycling conditions are you using, and what is your expected fragment size?
I have attached the image for both products. I run only a small quantity for confirmation. The rest was purified as a PCR product - to get rid of small nucleotides and enzymes, so, in general, I would have obtain a group of inserts and not a specific band to be used for ligation. I was expecting to get all kind of colonies, and could suggest the larger inserts would be an issue, but not the small ones.
Thank you very much for helping to figure it out
Sounds like you are trying to create a cDNA library? Blunt-end cloning will always be more difficult. One possibility- did you phosphatase treat your vector? Just wondering if phosphorylating your cDNA fragments is needed for ligation.
That looks pretty similar to the image from the kit protocol: http://www.thermoscientificbio.com/uploadedImages/Products/Molecular_Cloning/Kits/k2561_1.jpg
Which suggests your synthesis is ok.
You could of course check it by trying to PCR a couple of genes you know should be expressed: if you can pick those up then you're good to go.
This thus means you're probably having problems with your ligation. Have you specced your purified dscDNA to confirm it's clean? (260/230 ratio is important here: lots of PCR cleanup kits use guanidium -I think MN's kit does-, which is ludicrously inhibitory to downstream reactions). Also, make sure your vector is compatible with blunt-ended ligation: I'm assuming you phosphatase treat your vector to prevent re-ligation, but this might mean you need to add back phosphates to your dscDNA fragments (T4 polynucleotide kinase, for instance).
Yes, Amanda, I am looking for a specific sequence, most part of which I don't have, excluding two fragments that were used for primers design.
I asked Thermo should I do any additional treating to ds CDNA prior to ligation, and they say there is no need for it. I use pjet 1.2 blunt vector and it comes ready for ligation (ClonJet).
You are certainly right about blunt ligation. Along with that, I would expect to get a few colonies containing smaller inserts, but I didn't get even one.
I was thinking regarding the ds CDNA synthesis - could it be the second strand didn't work out or somehow the purification was unsuccessful (quality / product lost / partial degrading)?
John, may be you are right, and ligation didn't work because of low 260/230 - I got a ratio of about 1.0 for both after purification. Still I got two other bands - 1 kb and 1.5 kb- after gel purification with a lower ratio and got colonies for both. These were two other ligations I cloned along with ds CDNA to be sure I do not have troubles with Ligase, comp. cells, etc.
I hope you are right, and the synthesis was ok. It looks very similar to ss as well
Are you using the highest efficiency E.coli?
The only way to tell if second strand synthesis has worked is to run next to a sample of first strand only.
PCR will still work on first strand by the way, but obviously you won't be able to ligate. As long as you followed the kit instructions accurately, I'd guess it was ligation efficiency that was the issue, probably by contaminants carried over from cDNA synthesis. You could try to use a dilution of your cDNA for ligation.
Amanda, I used freshly prepared CaCl2 DHalpha with efficiency of 1X10^7, I guess it's ok, what are you saying?
The whole volume of first strand rxn is taken for 2-nd strand preparation. Next time i want to take a final product small quantity for digestion to be at the safe side.
Dilution allows to decrease contamination on one hand, but on the other hand we get less of insert. I was told it' better to fill up the reaction with DNA and to work with high volumes.
Thank you very much for your help
For library construction, I would buy competent cells that were at least 1x10^9 cfu/ug. If you have access to an electroporator, then >5x10^10 is possible.
Don't use more than 5ul to transform 50 ul bacteria or the efficiency drops dramatically. You could try ethanol/ NH4OAc precipitation (with glycogen or similar) to concentrate (and clean up) your cDNA.
I usually take 5ul to transform 100ul bacteria. I'll try to use a few dilutions next time starting with 5ul, getting rid of inhibitors first.
I guess both of you are right, it seems to be a problem with ligation and not with synthesis. Good news after all.
Thank you very much, to both of you
I agree with John...Test some primers, and do a clean up prior to this.
Ok guys, I synthesized another ds cDNA, purified it and got a cleaner product for both - oDT (700ng) ~ 10 kb+smear, and Random Hex (400ng) ~6kb+smear.
I am not sure about molar ratio - should I take an average insert size for calculation? (for blunt multiple insert ligation in pJet1.2 (50 ng/ul)
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