5 replies to this topic

[hbn]

Heey everyone,

I'm having a problem related to adapter ligation. I want to ligate adapters to both ends of a PCR fragment, like this:

5' Adapter - Fragment - Adapter 3'

The adapters are created from oligos which I have ordered from Sigma; I have annealed both of the single-stranded oligos to become the double-stranded oligo. After checking on gel, these look fine. The single stranded oligos have a 3' T-overhang and so should the double-stranded adapter.

The fragment is an amplicon from a PCR with a proofreading enzyme, the procedure which I have followed so far is the following:

1. PCR amplify the fragment
2. A-tail the PCR fragment
3. Phosphorylate the PCR fragment
4. Ligate the adapter to the fragment, having the adapter in a severe excess.

When I put it on gel after 'ligation', I just see a band for the adapter and amplicon seperately.
I feel like I'm doing something wrong here, anyone any suggestions?

[Michaelro]

Hi
Maybe you should try to perform PCR with primers fused to adapter sequences.
In this case, you could obtain PCR product with adapter sequences already in.
Good Luck
Michael

[hbn]

Michaelro, on 04 February 2011 - 10:54 AM, said:

Hi
Maybe you should try to perform PCR with primers fused to adapter sequences.
In this case, you could obtain PCR product with adapter sequences already in.
Good Luck
Michael

Thanks for the advice, but unfortunately that won't work; when you're using random fragmented genomic DNA (like is described in the protocol), you don't have any information of the sequences to design primers on...

[Maddie]

I'm doing Illumina libraries too but I am using the adaptors from the kit. I also have Sigma oligos to make my own but haven't tried them yet. I tried the P1.1 and P2.1 Sigma primers though and they do not work as well as the Illumina primers. But then who wants to buy 2 primers at 5,300$ ?? (for 100 reactions only, can you believe that?).
Anyway, I follow the protocol: blunt end, add A and ligation. I have papers with detailed protocols using PCR product, you want me to send it to you?
Are you sure that the adaptors didn't ligate? I mean you'll always get extra adaptors on a gel. Did you try to amplify your library?

[fisherning]

hbn, on 04 February 2011 - 10:23 AM, said:

Heey everyone,

I'm having a problem related to adapter ligation. I want to ligate adapters to both ends of a PCR fragment, like this:

5' Adapter - Fragment - Adapter 3'

The adapters are created from oligos which I have ordered from Sigma; I have annealed both of the single-stranded oligos to become the double-stranded oligo. After checking on gel, these look fine. The single stranded oligos have a 3' T-overhang and so should the double-stranded adapter.

The fragment is an amplicon from a PCR with a proofreading enzyme, the procedure which I have followed so far is the following:

1. PCR amplify the fragment
2. A-tail the PCR fragment
3. Phosphorylate the PCR fragment
4. Ligate the adapter to the fragment, having the adapter in a severe excess.

When I put it on gel after 'ligation', I just see a band for the adapter and amplicon seperately.
I feel like I'm doing something wrong here, anyone any suggestions?

Hi, how is ur project going now? Did ligation work? I thought your ligation did not work properly. U may check the A tailing process. For instance, you can use PCR product with A end using taq for ligation, and then do the adaptor ligation. If the ligation works, you may get ligated band on the gel.
I will do the adaptor ligation too for my gDNA. If you have time, can we have a discussion?

[hbn]

fisherning, on 24 October 2011 - 08:59 PM, said:

hbn, on 04 February 2011 - 10:23 AM, said:

Heey everyone,

I'm having a problem related to adapter ligation. I want to ligate adapters to both ends of a PCR fragment, like this:

5' Adapter - Fragment - Adapter 3'

The adapters are created from oligos which I have ordered from Sigma; I have annealed both of the single-stranded oligos to become the double-stranded oligo. After checking on gel, these look fine. The single stranded oligos have a 3' T-overhang and so should the double-stranded adapter.

The fragment is an amplicon from a PCR with a proofreading enzyme, the procedure which I have followed so far is the following:

1. PCR amplify the fragment
2. A-tail the PCR fragment
3. Phosphorylate the PCR fragment
4. Ligate the adapter to the fragment, having the adapter in a severe excess.

When I put it on gel after 'ligation', I just see a band for the adapter and amplicon seperately.
I feel like I'm doing something wrong here, anyone any suggestions?

Hi, how is ur project going now? Did ligation work? I thought your ligation did not work properly. U may check the A tailing process. For instance, you can use PCR product with A end using taq for ligation, and then do the adaptor ligation. If the ligation works, you may get ligated band on the gel.
I will do the adaptor ligation too for my gDNA. If you have time, can we have a discussion?

Hey fisherning,

Lastly, we abandoned the whole procedure and ordered a kit to do this (it was for sample preparation for Next Gen Sequencing).
It took too much time to get the protocol optimized and properly working.
Thanks anyway for your help!