Pretty much the same as last time. I’ll need some discussion…
Thursday’s ligation failed. So today I’m trying to adjust. Based on Bill Hooker’s suggestion, reducing the amount of ligase could be a solution so I’m trying that. I’m also increasing the ligation time between each adapter addition to give the reduced ligase time to ligate. Finally I’m adding a third reaction using the leftover DNA from Thursday (concentration of EpBR is 60nM and BpALS is 67nM). Check out the reaction below.
Below you will see the gel of the ligation reaction. It almost worked. I increased the contrast of the image to identify the successful part of the reaction.
The left most and right most lanes are 1kb ladders, and the 4 middle lanes are the ligation reactions. The 1kb ladder weights are (from the top): 10kb, 8, 6, 5, 4, 3, …
At 6.5kb there should be a band, and there is a feint one. There is another band just below that and I have no idea what that could be. The brightest bands are at 4kb (pALS) and 2.5kb (gel extracted pBR322). There is also another band just below that which I suppose is the other fragment of the pBR322 digestion, but I’m not too sure.
Anyways here is the image:
I’m almost there! Hopefully in a couple hours I’ll have unzippable DNA. But before we get ahead of ourselves let’s take the final step… together.
I’m setting up two reactions. The first is with some adapter DNA from a couple years ago, that I’ve gotten successful ligation results with. The second is the newer stuff I bought over the summer, that may not have worked all that well (inconclusive). Here is the protocol:
Yay, good digestion! Here is some useful information about which piece to gel extract. I’m always looking for this mostly for doubling checking purposes, but I need to figure out a place to store this so I can EASILY find it. For now all that matters is I cut out the larger band (top) and do gel cleanup. Onward and upward I suppose…
I extracted into 2 tubes and here are the final amounts:
- 88.7ng/ul –> 52nM
- 101.9ng/ul –> 60nM
The digestion is complete. There is really no way for me to verify the results so I just went ahead and did an enzyme cleanup and purified the DNA. Now I have:
- Tube 1 – 170.0ng/ul –> 63.75 nM
- Tube 2 – 178.3ng/ul –> 66.86nM
Once I verify the pBR322 digestion and gel extract (coming up next) I can set up the ligation reaction and finish up!
Since all the reactions yesterday worked, I purified the DNA and took some measurements in the nanodrop:
- Tube 1: 289.6ng/ul in 30ul –> 8688ng
- Tube 2: 259.8ng/ul in 30ul –> 7794ng
Since those yields look phenomenal, I decided to run my digestion of the anchor with BstXI. Once I gel extract my pBR322 digestion, I can run the final step, LIGATION! I’ve never completed this process in 2 days, and I hope I didn’t just jinx myself. Anyway, here is the digestion of pALS: