Did you nanodrop your samples before submission? My first thought would be that they're heavily contaminated with chaotropic salts (or organic solvents) from a bad miniprep, and/or have a very low concentration of plasmid. Sometimes even if you nanodrop a sample and it looks good, the plasmid concentration you're sending for sequencing is actually much lower than expected due to significant RNA/genomic DNA contamination.

My other guess would be that the primers you're using to sequence don't actually have binding sites in the plasmid. Sometimes that's due to weird cloning events like for example, inverse PCRing a plasmid and having mispriming events that delete part of it. Other times it's because the sequences you're basing your design off of are incorrect.

Either way I suggest restriction digesting a small amount of the sample and running it on a gel before sequence submission, sometimes that can give you an obvious answer as to where the problem lies.

I would concur with Alexandra above - the sequence read is only 240-odd bases long and there is considerable background even in the region with best quality peaks, suggesting the sequencing team had to increase the gain on the read to get anything usable at all.

Is the plasmid you are cloning into very low copy number? Are you certain that your mini prep kit solutions are in good condition (fresh ethanol in wash buffers etc)?

Any other information about how you prepared your plasmid, what the plasmid is, and so on would be useful for us to be able to assist further. Good luck!

1. sequencing is a one time binding event so your product purity is more important.

2. If your primer is not a common primer, u just designed for sequencing, try different. Common primer mostly wont bind and pick background it simple gives some NNNNNN

3. try to pick new and sequence again or re transform what u have cloned and isolate fresh.

Good Luck

Hi .. Alexandra and Robin and Karthikeyan Krishnamurthy , thank you for your responses to my enquiry. However , i have further questions to ask on this issue ;

Firstly, Alexandra, as regards your suggestion about '' heavy contamination of my plasmid samples with chaotropic salts (or organic solvents) from a bad miniprep, and/or have a very low concentration of plasmid and also a possible significant RNA/genomic DNA contamination of my plasmid sample'' , what do you suggest i do to minimize these contaminations ?

I used the Qiagen spin - column miniprep kit for my plasmid miniprep and i added fresh ethanol to the buffer PE which is used as a wash buffer , in the Qiagen Spin Miniprep Kit procedure. Please could you advise me on the right procedure to carry out my miniprep to prevent these contaminations in my sample again ?

I have also attached a picture of a gel showing the Diagnostic restriction digestion of the plasmids purified from the Transformation plates after the cloning experiment [Image 4 ---Diagnostic Digestion - 22- Aug-18 ]. The first lane of the gel plate is the molecular marker, the second lane is a linear plasmid which is the same size [ 5108 bp] as the linear plasmid used in the cloning reaction. Lanes 3 - 8 show samples of the purified plasmids from the colony plates of the transformation reaction , after the plasmids [ hopefully containing the the cloned gene insert ] were digested with the two restriction enzymes used to cut the plasmid, to linearise it for the cloning experiment. These plasmids should be 5612bp long. The diagnostic digestion was done to verify if the purified plasmids contained the gene insert. Specifically, lane 4 represents the plasmid sample which i used for the attached '' messy'' sequencing reaction.

I have also attached a picture of an agarose gel showing in lanes 4 -7 , other plasmids which were purified from the same Transformation plate as the sequenced plasmid. [Image 11 -Analytical gel for Linear pOPINF plasmid - 04 - sept- 18]. Lane 1 is the molecular marker and lane 2 is the linear pOPINF plasmid.

Further, i used the pOPINF plasmid for the cloning reaction. This plasmid is a high - copy plasmid. It is 5531bp long, and was cut and linearised using the Hind111 and Kpn1 restriction enzymes. The linear plasmid is 5198 bp long and the plasmid + gene insert should be 5612bp.

The primers i used for the sequencing are - the Standard T7 Primer [ forward primer - this was provided by the sequencing company - GATC Biotech ] and the Bglob-pA-R Primer [ reverse primer - this primer was synthesised on my request to the GATC Biotech ] . These primers are present on the plasmid backbone sequence and i expect that they should bind.

With these information and pictures, please could anybody give me further advise or suggestions on -

1] Possible causes and solutions to the Sequencing problem and

2] How to minimize ' heavy contamination of my plasmid samples with chaotropic salts (or organic solvents) from a bad miniprep, and possible significant RNA/genomic DNA contamination of my plasmid sample' ?

I look forward to helpful responses.

Thank you.

Uche

Hi Uche,

thanks for the extra information. Right now the first thought I have is that the colonies chosen do not contain the vector you are looking for - the linear band in lane 2 is quite clear. Lanes 4-7 if this is supposed to be the same vector with insert excised using the same enzymes as the linearisation in lane 2 should give a band the same size as lane 2 with a ~5-600bp long insert band. Neither of these is the case, suggesting that you are trying to sequence something that is unlikely to feature your insert or maybe even the expected priming sites.

This can happen to anyone, so don't get too upset. Firstly - are you sure that your plates have been made with fresh antibiotic and are not excessively old? Do you have other controls for the transformation (i.e., uncut vector, ligation without insert, cut vector without ligase etc. ; untransformed cells only on the antibiotic plate) - as if there is intact DNA contamination of any buffer components, vector carry-over, or the bacteria already carrying a vector this could also give spurious results.

I would probably start by preparing new stock of linear vector and insert, purifying these, verifying that the purified DNA is o.k for both parts, and then prepare new ligations from scratch rather than trying to rescue what you already have as this would probably waste further time and reagents.

Best of luck!

Hello Robin.

Thank you for your response.

I have attached the picture of plates from my Transformation reaction , after the cloning reaction. Like you have suggested , i I have also repeated a fresh restriction digestion of the pOPINF plasmid and have attached a picture of the agarose gel.

The gel extraction of the linear plasmid products from the fresh restriction digestion only gave about 11.2 ng/ul, this was eluted in 30ul of distilled water..The purity ratios of the recovered linear plasmid are 1.90 [ lamda 260/ lamda 280 ] and 0.01 [ lamda 260/ lamda 230]. I want to use this recovered linear plasmid for a fresh cloning and transformation experiments.

In your opinion, is the recovered plasmid pure ,What do you advise ?

Further, what do you think about the plates from my Transformation experiments ?

Finally, could you please suggest to me and alternative molecular biology technique to ensure recovery of a high percentage of my pure, linear plasmids ?

Thank you.

Uche

Hi .. Robin..

Sorry, i forgot to explain the plates of my Transformation reaction, on the attached picture -

The first plate , at the top , is my '' positive control'' .

The second and third plates, below it , are colonies which grew from cloning reactions using 1 :3 and 1 : 5 vector : insert ratios respectively..

Thank you.

Uche

Hello.. Alexandra.

As you suggested in your answer to my earlier question, how do i ensure that my purified plasmids would not be heavily contaminated with chaotropic salts (or organic solvents) from a bad miniprep, and/or have a significant RNA/genomic DNA contamination in future. What strategies or measures can i take to avoid this ?

Uche

First off, how many ng of your plasmid are you submitting for sequencing? I would recommend trying different primer sets that would also theoretically read into your region of interest.

Hi Aleksey..

Thank you for your response. I submitted about 46ng /ul of this plasmid. Plasmid + insert should be about 5,601bp long.

Further , what different primer sets would you recommend ? The cloning vector is the pOPINF plasmid.

Uche

That is ok 46ng/ul plasmid conc. How much is there in total conc (it should be more than 450) depends on the core lab the need of total plasmid various.

PCR sequencing u need less for plasmid u need to submit more

If u have made mistake there u get peak messy

Check with the core lab

Hi Uche,

Looking at those plates my alarm bells are ringing.

1) there are satellite colonies everywhere, and what are probably the principle colonies are very large. Either the antibiotic is being added to the medium when it is too hot (so the effective concentration is reduced), or the Amp stock isn't fresh, or it is insufficiently concentrated - or your fridge isn't cold enough, or they were over-grown. I would start with the antibiotic question. Are you using Ampicillin 50 micrograms / ml? If you have the option of switching to 50microgram / ml Carbenacillin or a higher concentration of Amp in the medium (100 or 200), it would be preferable. Secondly, pouring the plates - when the agar medium is liquid, using a water bath or similar, cool the medium to around 50 degrees centigrade maximum before adding the antibiotic, then pour and cool the plates as fast as possible, otherwise the antibiotic will degrade. Plate the bacteria as late as possible, and if you have the option, grow at less than 37 degrees (for instance - 35.5 degrees) overnight. This will mean that the colonies are very small in the morning, but you can then continue growing them during the day at 37˚ and watch them until they are large enough to pick late afternoon without satellite colonies growing up and risking contamination of cultures. Otherwise, you can get overgrowth of principal colonies with satellites growing around them during the space of a normal overnight incubation as local Ampicillin is degraded by secreted beta lactamase. Final possibility is that the large colonies are contaminants and not your E.coli at all.

2) you have a positive control and two ratios for the ligation. Do you have an empty vector religation control without insert? This should be performed with every ligation, without exception to ensure that you are not growing empty vector colonies on what you believe to be ligations. If you want to save plates, plate both ligation and ligation control on the same plate with a line drawn down the middle to separate where they are streaked.

Sorry to sound harsh, but although this started as thread about sequencing problems, everything you've shown so far indicates that the problem is much further back in the sequence of processes. Until these issues have been resolved, discussing the sequencing reaction is equivalent to consideration of deckchair layout on the Titanic.