I am trying to insert four different sequences, indivually, in a backbone. So, I will have four different clones all with the same backbone. I am using a modified pcDNA3.1(+) plasmid that has had its BamHI and HindIII sites deleted. It is 5400bp. My inserts are approximately 1600bp for two of them and 1700bp for the other two. Three of my inserts are geneblocks. I digested all my inserts and my backbone with XhoI and EcoRI. I first digested my backbone with my restriction enzymes then I gel extracted. I did a PCR amplification on all of my inserts(one of which is coming from another plasmid). I did a gel extraction, then I digested with my restriction enzymes, and then did PCR purification. Then I ligated, in a three to one ratio, 3ul of backbone to 9ul of insert in a volume of 20ul. The concentrations for my backbones were about 24ng/ul and the inserts had 70-100ng/ul approximately. I have tried both T4 ligase(incubate overnight in the fridge at 4 degrees) and Quick ligase(incubated at RT for 5min). I think used 10ul of ligation product and transformed homemade DH5alphas, another labmate of mine did get good results with them. I have tried Stbl3s in the past but ended up getting smears when I ran the clones on a gel, I'm pretty sure its the endA+ messing it up. Oh, I have also tried putting the geneblocks into a pLVX backbone, but that also failed. I am thinking of trying Transformax cell this time. Any thoughts or advice would be greatly appreciated. Oh, here are the sequences(V2.1,2.2,3.1 are the geneblocks):
You have not told us the problem. Are you getting no colonies at all after transformation? Or are you getting colonies but none have inserts?
You need to do appropriate controls to determine 1) are your competent cells good enough for cloning purposes 2) is your ligase still good 3) is your ligase buffer still good (ligase buffer goes bad if freeze-thawed a number of times or stored at room temp for extended times).
If you are getting no colonies you need to confirm that you have good vector backbone after your manipulations.
By the way, you can circumvent the EndA problem from STBL or other EndA+ strains by NOT treating with RNase. The free RNA in the mini-rep will inhibit EndA activity on your DNA during the restriction digest. Just the run the gel a bit longer and all the RNA will be far away from your bands.
Hi Michael J. Benedik ,
I apologize for not being more clear. I had a few tries where I got no colonies after the transformation, I was using homemade DH5alphas, but for the most part I got colonies on my Ampicillin plates. My issue stemmed from when I was validating that I got my clones. After doing mini-preps on selected colonies, I was cutting with my restriction sites and then running on a gel. I was expecting to see two bands, my insert and backbone. If I did then I would send out for sequencing that specific mini-prep. I was not seeing those two bands, I was seeing either streaks or bands not at my expected lengths. My PI eventually just had one of the postdocs get the clones. But I think I know what I was doing wrong. After looking back, I think I was using bad starting vector. I wasn't paying enough attention when I was specing them, I was just looking at the yield only and not the graph and 280/260, 230/260 ratios. Looking back I did not have good mini-preps of those empty backbones. I think that was where most of my issues came from.
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