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DNA sequencing after ligation! HELP!

PCR primers DNA sequencing

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8 replies to this topic

#1 Bpaul

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Posted 26 August 2012 - 05:35 PM

Hi All

I am pretty new in molecular biology and hence this question!

So i got the ligation to work and then sent it for sequencing. I used the PCR primers (primers i used for the PCR reaction!) for sequencing! So my protein is 210 amino acids long. But i will use a random protein sequence to ask my actual question. So consider this is my protein sequence...

MDLDIKQSQL AATNRRHGKW DEWSDKRESR VWKTDCRIFG

I used both forward and reverse primers (as seperate reactions!) for sequencing. I was hoping to see the entire protein sequence in my DNA sequencing result! But instead i saw the results like this..

Reaction no.1. With Forward primer:

The resulting sequence showed match with my protein sequence from AATNRRHGKW DEWSDKRESR VWKTDCRIFG and the ones before (N-terminal ones!)that was something else!

Reaction no.2. Reverse Primer:

The results showed match with my protein sequence MDLDIKQSQL AATNRRHGKW DEWSDKRESR and the end portion was something else!

Both the reaction mixtures were set up from the same DNA tube! so when i look at the bigger picture i have my insert! But i was hoping that the results will be coming out as the entire insert and not like what i got! So my question is... is this normal? Is this how it should be?

Sorry about this very stupid question! But please help! Will be greatly appreciated! Thank youPosted Image

#2 Curtis

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Posted 26 August 2012 - 11:43 PM

Sequencing machines are not usually able to read the first 10 to 20 nt of your sample. This is from both sides, 3' or 5'. That is why most people do not use the PCR primers for their sequencing. Instead, you can use the primer of your plasmid's promoter. Every expression plasmid has a promoter right before the MCS site. If your PCR product is not long (less than 1000 bp) you must sequence with the primer of that promoter (usually T7, T3 or CMV), if it is longer than that you can use multiple primers. You can design primers that bind to the regions inside the PCR product and ask the company to sequence your plasmid with those.

Moreover, when you send sample for sequencing you need to always talk about DNA sequence, strand, 3' and 5'. Using amino acid or protein sequencing in such conversations are not professional.

#3 ascacioc

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Posted 27 August 2012 - 06:24 AM

I can add that we do not send ligation reactions for sequencing: we first transform the ligation into a cloning strain bacteria i.e. DH5alpha, XL1Blue... then miniprep the plasmid and send that for sequencing. What use is to PCR the ligation and then send it for sequencing? Of course you will PCR your insert, ligated or not. It is a waste of sequencing labels (7 € per sequencing reaction) to sequence something that you are not sure you can amplify as a plasmid. What would happen when you transform it and it did not work? Or it is not ligated at all?

Andreea

#4 lyok

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Posted 27 August 2012 - 10:17 AM

Sequencing machines are not usually able to read the first 10 to 20 nt of your sample. This is from both sides, 3' or 5'



Why arent they able to sequence the first few bases?

#5 zodiac1505

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Posted 27 August 2012 - 11:50 AM

I agree with Andreea. It is better to make mini preps from the transformed colonies and then send them for sequencing with primers for the promoter used in the plasmid. We even used to verify the inserts with either restriction digestion or colony PCR and only send in the positive clones for sequencing.

#6 ascacioc

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Posted 27 August 2012 - 11:54 AM

Of course, totally agree with Zodiac1505: first do a check of the insert in the colonies. One sequencing reaction is like 7 euro (here in Germany), I don't know for other countries. It is damn expensive while colony PCR is much cheaper, so is restriction digest.

Andreea

#7 mdfenko

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Posted 27 August 2012 - 01:35 PM

Sequencing machines are not usually able to read the first 10 to 20 nt of your sample. This is from both sides, 3' or 5'



Why arent they able to sequence the first few bases?

with sanger sequencing, even after cleaning, some unincorporated dye terminators remain. these create dye "blobs" which will obscure early sequence results. more efficient cleaning will allow you to read closer to the primer (as close as 5 bases away).
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#8 Bpaul

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Posted 27 August 2012 - 07:52 PM

I can add that we do not send ligation reactions for sequencing: we first transform the ligation into a cloning strain bacteria i.e. DH5alpha, XL1Blue... then miniprep the plasmid and send that for sequencing. What use is to PCR the ligation and then send it for sequencing? Of course you will PCR your insert, ligated or not. It is a waste of sequencing labels (7 € per sequencing reaction) to sequence something that you are not sure you can amplify as a plasmid. What would happen when you transform it and it did not work? Or it is not ligated at all?

Andreea


Hi

Sorry...! But when i said i sent the ligation reactions for sequencing i meant i transformed them, minipreped it and then sent it for sequencing :-).. Sorry about the confusion! I did RE digest the minipreps to see if the inserts are there! And it was there too!... I talked to someone from my lab now and they also said the same as the above answer... that the sequencing machines wont read the first few bases!! Thanks everyone! :-)..

#9 Bpaul

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Posted 27 August 2012 - 07:53 PM

Sequencing machines are not usually able to read the first 10 to 20 nt of your sample. This is from both sides, 3' or 5'. That is why most people do not use the PCR primers for their sequencing. Instead, you can use the primer of your plasmid's promoter. Every expression plasmid has a promoter right before the MCS site. If your PCR product is not long (less than 1000 bp) you must sequence with the primer of that promoter (usually T7, T3 or CMV), if it is longer than that you can use multiple primers. You can design primers that bind to the regions inside the PCR product and ask the company to sequence your plasmid with those.

Moreover, when you send sample for sequencing you need to always talk about DNA sequence, strand, 3' and 5'. Using amino acid or protein sequencing in such conversations are not professional.


Thank you! This is the same answer i got from someone in my lab! thank you! :-)





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