tales from the ols building Episode 1: Cloning

My cloning worked!!! 
It took a good 6 months, then briefly on and off for a year and a half, but I'm happy to report that apparently persistence works even when you've completely given up all hope!
If you're like me with your cloning attempts, you've gotten great amplification with your insert, you've already got the vector ready to go, AND you've probably even successfully cut your vector (AND insert!) with several restriction enzymes AND THE BANDS look great, HOWEVER..dun dun dun!!! Then comes the dreadful part. The gel extraction!
Aghh I kept loosing everything here. Or even at the PCR cleanup step. Was it the water? The pH of the buffer? Old EtOH? Come on internet, someone has to have a solution to my cloning woes!!! So many questions, so little time, and nothing's working!!! UNTIL... I had tried so many times and enough time had passed for me to really give up on the project completely that when I thought about doing this again I had a renewed sense of what the heck was going on here.

1. DO NOT LISTEN TO NEB!!!
Ok, NEB has some great tools and advice AND they are actually awesome, however, they recommend completing a digest in a 50uL reaction volume. Doing this was the absolute worst. Especially if you're already starting with a small amount of DNA. It only makes sense that you would want to keep the molecules as tightly packed/close together as possible to increase the chances these things will run into each other. This may sound like common sense to a veteran clone-master, but to this gumby I was just following the instructions!  This was my main problem.
SOLUTION: KEEP ALL YOUR REACTION VOLUMES (FOR PCR PURIFICATION, GEL EXTRACTION, & LIGATION) ANYWHERE BETWEEN 10-20uL. If you can't do this, then your starting material is already too diluted & you NEED MORE COWBELL! This brings us to #2:   

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2. START WITH AT LEAST 1UG DNA (in 10-20uL) GOING INTO THE DIGEST.
This is for both your insert and vector. You should definitely PCR purify the insert. If you're having trouble with loosing material, combine two 50uL PCR reactions together for the purification and elute with a 20ul reaction volume. This way you will have a super concentrated stock that you can pull 1ug from to complete the digest and if it doesn't work you don't have to repeat your PCR again. 

3. rSAP treat your vector!
Once I started increasing my concentration of DNA, I thought everything was going great, however I would get all the way to the transformation and then verify my clones and they would all be vector only. People actually told me phosphatasing the vector was not necessary, but if you rSAP treat the vector the chances of it re-ligating in the face of 3X more insert than vector are pretty low. Also the rSAP works in all NEB buffers so just add 1uL in after your digest (but before gel extraction) for 30min to an hour at 37C and you're good to go. If you're going to do the gel extraction you don't need to heat inactivate it.
  
​4. The DREADED GEL EXTRACTION!
Make sure you're loading AT LEAST 1ug DNA per sample per well- this is why you want 10-20ul total reaction volume for your digest. You can't fit 50ul (60ul + loading dye) into one lane, so you'll have to pool two gel extracted pieces together. While doing this does increase your yield, I guarantee the quality will be too poor for you to do anything with it downstream. Once more, you will get to the transformation and you'll have nothing but vector. If you're expecting more than one band to form after your digest and you've loaded 1ug AND you're only cutting out one band from the gel for the ligation, you're going to be loosing a lot of the original DNA. You need to make sure to start with enough! Additionally, once you've extracted from the gel make sure to elute into 10ul, it's as simple as that. You will need concentrated vector and insert DNA to keep your ligation reaction at 10ul total volume. ​

After taking these 4 steps into consideration, I was able to run through the protocol all the way to the transformation; then I selected 6 clones, and unlike previously this time the digest told me they were all right!  I sent all 6 for sequencing (because of being so skeptical that it had actually worked) and they were all correct!

If you would like a copy of my full protocol from primer design to sequencing please leave a comment below or contact me here. I would be happy to share my protocol with you. Hopefully these tips will help save you the frustration I went through!