What restriction sites are you using? How much overhanging sequence is present between the end of your oligos and the restriction site? How many colonies do you get on your control ligations (vector without insert)? Have you quantified the amount of insert in your ligations?

Incidentally, Promega's pGL4 series of vectors are much better in terms of signal to noise compared to pGL3, so I would recommend switching to the most current vector system.

What restriction sites are you using? How much overhanging sequence is present between the end of your oligos and the restriction site? How many colonies do you get on your control ligations (vector without insert)? Have you quantified the amount of insert in your ligations?

Incidentally, Promega's pGL4 series of vectors are much better in terms of signal to noise compared to pGL3, so I would recommend switching to the most current vector system.

Hi Daniel, I'll try to answer your questions:

1. Kpn I - 5' and Hind III - 3'

2. 4bp

3. It seems the same number as the insert ligations (I know...)

4. Yes, I've used 2 ratios between insert-vector (1:1 and 3:1)

I don't think it's a problem with enzymes because when i cloned smaller pieces of this promoter (e.g. 2500 bp upstream) it worked..and I used the same restriction sites (the reverse oligo was the same!).

Hi Valerio. Since the strategy worked for a smaller piece, that eliminates any concerns with the enzymes (digestion, ligation). So here are some possible reasons you may be running into problems:

1) Your larger fragments contain internal sites for KpnI or HindIII that you did not expect to be present (SNPs can cause this). If you haven't already done so, make sure you digested material runs at exactly the expected size.

2) In general, efficiencies of ligations decrease with insert size. When the vector and insert are similar in size OR the insert exceed the amount of vector, it is critical to keep your ligations to a 1:1 molar ratio. Also total DNA amounts should not exceed 100ng in a 10-20 uL ligation reaction. If your cloning efficiency was just OK with the smaller 2.5kb fragment (you got positives, but you had to screen a lot), you'll have to improve the quality of your digested vector prep. Can't tell from your reply how much of difference in # of colonies there was between positive and negative ligation mixes in your success cloning of the 2.5kb promoter. Another potential issue is that DNA quality is not always good for some kit-based isolations of larger DNA fragments. Check the DNA on a Nanodrop to make sure you don't have contaminating guanidinium salts (high signal at 230nm and below), as these will inhibit your ligase.

3) Your larger promoters may harbor a sequence feature that is interfering with plasmid replication in E. coli. Have you checked for any special sequence features, including large CpG islands, in these larger promoter targets?

Tank you so much Daniel! I'm pretty sure that there are not restriction sites into the sequence as the running profile is correct. I totally agree with the sentence 2 and I can tell that my gel isolation kit is very very old so it's possible that my DNA has a low quality (I will check out). I didn't checked for CpG islands but I have to do it as soon as possible. I really appreciate your answer.

For what it is worth, I've also found that unless the DNA concentration is at least 10ng/uL, it is very hard to get good quality ligation products. Probably a function of poor DNA quality and inaccurate concentration estimates. Luckily none of your fragments are so large as to render plasmid replication intrinsically difficult (>20kb, for example). So I'm sure with a little perseverance, you will get these promoters cloned.

Hi Daniel, just to give you an update, I've checked my sequence and I found a CpG island (from 7766 to 7979). Do you think it can affect the cloning?

Anectodally, yes. While I have been able to get promoter clones that encompass CpG islands, I think that there are cases in which these sequences can impede plasmid replication and cause problems with obtaining positive clones. Might be worth designing a new PCR primer that will omit this region, just to test whether it is the source of your problem. You could always try reintroducing the sequence a later step (esp. since it is quite small).

Incidentally, what E. coli strain are you using?

DH5alfa...

DH5a should work. If you can get your hands on a TOPO-based cloning vector, you might give this a shot. In some circumstances, this works much better than T4 ligase-mediated cloning.

The last try was on a Strataclone vector that is similar to TOPO-TA, I amplfied my sequence with a takara polymerase to have overhanging extremities, when I transformed cells provided by the kit I had 50% white colonies but seems to be negative for the insert...

How did you screen for the presence of the insert? Colony PCR can yield false negatives sometimes. With this large an insert, you should just boil the bacteria to lyse and load the samples on a gel to look for slow migrating plasmid (no PCR, no purification).

Both with colony PCR and with plasmid isolation (miniprep) followed by a restriction assay

I would suggest you retry your PCR amplification at this stage. If you can get a clean, single product in the PCR reaction, try the TOPO-based cloning without gel isolation (just a simple PCR column cleanup to remove oligos). It is important that you don't add excess insert (otherwise each end of the vector will react with a different insert product and the vector can't close). If in doubt, try serial dilutions of the insert (ideally 1:1 molar ratio with vector).

Tank You