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Cloning protein of interest into lentiviral vector - help - I have no experience

molecular cloning plasmids

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#16 OldCloner

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Posted 09 September 2019 - 01:11 PM

Like Bob1 I recommend that you sequence the entire insert; even the best proofreading polymerases can and do make errors, especially if there is anything funky about your insert (long single base repeats, or high or low CG content, etc.). In our lab it was considered a requirement to sequence anything cloned from PCR products. Bob gave you good advice on designing primers. If you become an "Old Cloner" you might stretch the intervals to 500-600 bases, but for now 300-400 is good. (I had the advantage of working in a sequencing lab, did my own sequencing and got really good at estimating how far my reads would go. I also ordered the internal primers, with the amplification primers, at the start of the project, to save time and shipping costs.)  It is good to sequence at least one clone before continuing to your final product; keep the other plasmids in case you do find an error, and you can check another one if your first one has an error. Also I would check to make sure you got the appropriate hybrid recombination sites for the Gateway reactions. Congrats on getting this far as a first time cloner!


Edited by OldCloner, 09 September 2019 - 01:19 PM.


#17 Natalia KM

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Posted 09 September 2019 - 07:46 PM

Thanks! Let?s hope I get good data from these experiments!

Sorry I didn?t get what you meant by ?appropriate hybrid recombination sites for the Gateway reactions??

Is there any real difference between sequencing primers and other primers such as qPCR primers for example? Is there anything I should be aware of that needs to be done differently when designing sequencing primers?

Thanks again for all your help - was a great learning curve! My next project is to design crispr knock out guides and clone into appropriate lentiviral vectors so will start a new post on that in a few days! Any help on that from yourselves and anyone else would be great!

Thanks

Edited by Natalia KM, 09 September 2019 - 07:55 PM.


#18 bob1

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Posted 10 September 2019 - 08:07 AM

What OldCloner meant was that there are sites on the plasmid - on either side of your insert - that are the sites that the gateway cloning system uses to get from one plasmid to another. These are usually designated AttR or AttB or something similar. If these sites are mutated, the gateway system will not work, so he was saying that you should confirm these sequences are correct too.

 

Primers are primers - the ones you use in real-time/qPCR are no different to those for regular PCR. If you are using a Taqman system then there are also the probes which are different and not the same as primers in that they insert between the primer sites. Basically however, they all boil down to the same thing - an oligonucleotide sequence that binds DNA. There are no special considerations for sequencing, just do the regular annealing temp/GC content things. You don't need these in pairs - only one primer per reaction for seqencing, as I am sure you already know. 



#19 mdfenko

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Posted 10 September 2019 - 11:18 AM

One small point regarding primers for sequencing. With Sanger sequencing, longer primers are recommended. For pcr, primers of ~20 bases are recommended. For sequencing, unless the recommendation has changed in the past 2 years, primers of ~30 bases are recommended. This makes them a little more specific and a little less prone to slippage.

That being said, I used to sequence successfully with pcr primers.

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#20 OldCloner

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Posted 12 September 2019 - 04:26 PM

Well, here's a different opinion: I usually design sequencing primers of about 20 base pairs, and try to get a 50 to 60 percent GC content. Avoid repeat areas like "AGCAGCAGCAGC" and similar. Sanger sequencing usually involves an annealing temperature of 50 degrees C, so the Tm of your seq primers should be a bit higher.

 

It is true, however, that using longer primers for sequencing helps you get reads that begin closer to the primer.  You may have noticed that your chromatogram begins with some poor quality jumbles of colored peaks that you have to edit off.  With longer primers, even the shortest fragments generated in the PCR rxn are longer, so they are not as prone to getting lost in the ethanol precipitation step that precedes electrophoresis.(That is part of the reason the beginning of chromatograms are so poor)  But if you have a lot of overlap between your chromatograms, you usually don't have to worry about reading close to the primer. And yes, you can often sequence with a single primer originally designed for PCR. If you are not too familiar with how Sanger sequencing works there are some decent videos on YouTube.

 

Check your Gateway manual; it will explain how the recombination sites work and how you get a hybrid recombination site when it is done. The resulting att site is a little different than either of the two sites you started with. The manual should show the original sequences and the resulting hybrid. Manuals are great educators! If you don't have a paper copy, you can find it on-line.

 

The only difference between PCR primers and sequencing primers is that for PCR, you are often adding "tails" at the 5 prime end: tags, restriction sites and whatnot, so they wind up being quite long, sometimes.  Also you have to take into account, in designing them in pairs, that they don't stick to each other to make the dreaded "primer dimer."

 

As for qPCR, primer design can be a bit trickier, depending on your template and purpose. Not so much the length or nucleotide composition of the oligo itself, but where in the target sequence you put them. And what  protocol you are using for detection: Taq Man or Sybr Green are the most common methods. It is too complicated to get into now, but if you ever need to do qPCR you can ask for help here with your specific project. Or read manuals that come with the kits- often they help a lot.

 

Good luck!


Edited by OldCloner, 12 September 2019 - 04:45 PM.


#21 Natalia KM

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Posted 17 September 2019 - 01:37 PM

Thank you so much for everyone's advice! I would be really grateful if everyone could have a look at my crispr post as well so I can pick on your brains! Thanks







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