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I'm a complete newb at doing cloning, this is my first time. After I cloned my stuff, I Sangered it, and the results were a bit perplexing, so just wondering if an expert can shed light on this. My goal is to obtain the sequence of a set of adapters in a WGA kit so that I can design a method to remove it from my PCR products. Please bear with me for the long description, and thank you if you get to the end and have some suggestions for me.
1. My genomic material is random primed and amplified with this unknown adapter, so all my DNA fragments (150bp-1kb smear) now should have this adapter sequence at the ends. They should be blunt from the PCR amplification step. These DNA are in EB, purified using a MinElute kit.
2. I want to Sanger these fragments to find the adapter sequence, so I used the Invitrogen TOPO TA cloning Kit.
3. I know that you need an A overhang in order to do the cloning. The A overhang on my PCR products probably already degraded, plus I don't know which polymerase is used in the WGA kit, so I can't for sure say that an A was added in the first place. So, I used an End repair protocol, followed by a Klenow protocol to add an A overhang to my PCR products (I followed the protocol used to add A overhang in Illumina library prep protocol).
4. Now my products are supposedly A'd, I use it per instructions in the cloning kit, using heat shock method. I used TOP10 e. coli that came with the kit. I also included a PCR control that came with the kit, and a pUC19 control.
5. After growing plates overnight, I only got ~10 colonies for my sample, and lots of colonies for both controls, which indicates that my kit and technique is okay. I went ahead and picked the colonies from my plate anyways (8 of them), and did a PCR using M13 primers followed by gel to check insert size. They had various different insert sizes, ranging from ~200 to 800bp. I then grew these up in LB, purified the plasmids using Qiagen MiniPrep, and sent them off for Sanger using M13 reverse primer.
6. The result of the Sanger was that 6/8 colonies had exactly the same sequence (I checked using Blast), and only 2/8 had unique sequences. Because of this, I have low confidence in identifying my unknown adapter sequence.
My questions are:
a) Why did my plate only grow so few colonies? I'm pretty sure I added in ~300ng of DNA into the ligation step, which is a lot, and I checked my sample with a gel before, so I know the DNA is not degraded.
I suspect that the A overhang addition didn't work well. If that is the case, what protocol should I use to add A overhang to purified genomic product to ensure higher cloning efficiency?c) Any suggestions on why the Sanger sequences are so duplicated between 8 colonies?
Thank you so much!
Angela
