The confirmed colonies you have were checked by the same REs prior to sequencing?
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Is this a kit based extraction or a crude phenol-chloroform extraction?
When DNA concentrations are low, you can precipitate your DNA out with 10% NaOAc/EtOH for 30min to O/N at -80C. You also add glycogen if your ethanol solution if your DNA is being difficult.
Edit: Oh the typos...
The ratio refers to the amount of vector to the amount of insert, i.e. 1:1, 1:3, etc...
You have used this kit with the provided control and are sure that it works?
I have used sticky end based TOPO kits and have had varying success depending on the gene. Double check and make sure you are correctly calculating the concentration/ratio of your vector and insert. If varying the ratio of the vector:insert doesn't work, I would consider increasing the TOPO reaction time.
You could feed your insert though a secondary structure prediction program and see if it is forming a weird structure that is impeding proper ligation... but this is probably has nothing to do with your problem.
How many colonies did you test?
How are you transfecting your cells? Are you doing a transient transfection, waiting two days and then transfecting again?
Do you linearize your plasmids when you transfect your cell with multiple plasmids. If not, I would at least linearize the LacO plasmid. That guy is huge and the cell may have some difficulty taking it up.
I would start by transfecting your lowest transfection efficiency plasmid, selecting for it and then proceeding with the others. How are you selecting for your plasmids? Does each plasmid harbor a different mammalian resistance or are you doing downstream assays to check the expression of each plasmid?
You could nanodrop your PCR sample or use a DNA specific fluorometer for an exact reading; however, using HindIII for densitometery is an alright method.
Increasing the reaction time could potentially help you. You may just run the reaction until the enzymes are dead, but it shouldn't hurt you overall.
Sometimes colleagues prepare the wrong concentration of antibiotics. Double check and make sure you are adding the appropriate concentration.
Can you share the gene you are trying to TOPO?
Edit - One more question... Can you cut out your gene from your other plasmid using a blunt RE? Phusion polymerase is amazing, but maybe something funky is going on.
Sounds like your DNA preparation was not optimal. I would try to make new cDNA from any remaining RNA you may have. I have had huge fluctuations with my HPRT1 values when my RT reaction went hay wire. HPRT1 Ct values of 25-30 makes it almost impossible to quantify your data.
Try your HPRT1 primers on another sample that you know has worked perfect in the past. Maybe freeze-thaw of your primers has caused degradation, but I doubt it. Also run each HPRT1 reaction on a gel and see if you see sharp bands.
I would keep the amount of RNA you use for cDNA synthesis constant. It shouldn't matter as long as you only analyze your data from the controls run with those increased RNA concentrations, but it would make it difficult to quantify results from previous experiments (i.e. checking expression of a gene from this experiment, to a previous experiment) - does that make sense?
It is possible that your RNA has degraded over time. Is there anyway you can get fresh DNA?
Try another reference gene and see if you get higher than normal Ct values?
When you generated your new insert, did you purify it or does it still contain the DNA polymerase. Could it be possible that your plasmid is recircularizing with the T4 ligase and then the polymerase is filling in the gap? The ligation would be energy favorable, but would your polymerase function?
We store them for up to 14days. After that, weird things can happen.
Has you lab ever considered switching to a crude phenol-chloroform extraction method to save money. This method can be done when trying to isolate ligation products and the midiprep columns can be saved when preping large quantities of the sample.
Edit - Storage will probably depend on your E. coli cell line. This is in reference to DH10beta. I haven't tested on additional cell lines.
When you go to endnote in word > search references > the external endnote program opens > do you see a drop down menu with formatting styles? I have version X5. You can also find this info in edit > output styles.
There are a lot of different detergents out there. I think you should search highwire.stanford.edu with you key words, "your cell line, cell lysis buffer, homogenization" or something along those lines. Find a good publication that has beautiful westerns and mimic their methods.
If your electrode dried out (solution precipitated), you would have an accumulation of salt on your electrode. The hot water increases the solubility of the salt, allowing for proper conduction between your pH bulb and solution.
No. On a A260/280, pure RNA is close to 2.0 while pure DNA is close to 1.8. Your reading indicates the presence of a contaminant. Probably protein, salt or a buffer left over from your RNA purification.
Depending on the downstream application of your RNA, you should consider doing an isopropanol purification.
Edit - Depending on how you spec your sample, (nanodrop, cuvette, etc.) make sure that the utensil is clean. I have been in labs that dishwash cuvettes. Soap scum on the cuvette will interfere with your reading.
I don't have a protocol that is easily accessible, but I am sure you can google one or someone on here will be happy to contribute one.
This experiment would essentially be an IP. You would obtain a cell extract and add random DNA oligo's that are radiolabeled. Incubate the mixture to allow proper DNA-Oligo binding. Wash your sample and then add a specific antibody that can recognize your protein. Incubate an pull down using magnetic or agarose beads. You could then run a gel and detect any radioactivity on autorad film. If you see a band(s) you would know if your protein or multiple proteins are bound.
Does this make sense?
If you are extracting blood, it looks like your protein precipitation buffer is no longer viable. The red color is due to the protein hemoglobin being present in the solution. I would double check your protocol and make sure that you are allowing for proper precipitation of your protein and also check to see if your precipitation buffer is coming out of solution.
Are you centrifuging your samples adequately each time? Is it possible you are overloading the kit with blood each time?
As far as the brown pellet, it looks like you still have cellular debris in your sample.
Are you using fresh blood and/or are you properly resuspending it before beginning the isolation? If it is older blood, it may contain fibrin which can induce clotting and will make it more difficult to adequately remove all protein.
You can remake the cDNa and compare it to your previous run. Just make sure you use a control and that you keep your conditions consistent. If you are worried, run a couple of different normalizes during each qPCR and this will help convince you that your cDNA is consistent.
Are you saying that you want to clone your gene of interest into a vector that will allow for suppression of your gene?
Edit - That is confusing. Are you saying that you want to clone your gene into a vector, in the REVERSE direction with the hopes that it will hybridize and suppress your mRNA?
This isn't as simple as you would imagine. Researchers pay a decent amount of money for companies to manufacture antisense oligos for gene suppression. Usually when you request antisense oligo's, the manufacturer will send you multiple different plasmids containing different oligo sequences that could potentially disrupt gene expression. As you would expect, it is difficult to determine the correct anti-sense sequence that will disrupt gene function and multiple need to be used to determine which sequence can convey the greatest degree of supression.
If you are serious about doing this yourself, you should scan all the relevant literature and try to find a group who has successfully "knocked down" your gene of interest and request the oligo sequence that accomplished this. You may even be able to request a small sample of their plasmid if the lab is feeling generous. I honestly wouldn't attempt this myself, unless I had a significant amount of free time and even then I do not think it is worth the time to develop these myself.