Berglund:Cloning: Plasmid ligation
Ligation and Transformation
Cut PCR product (same cuts as the plasmid)
T4 ligase and T4 ligase buffer (commmercially prepared)
1.25ml of 4M KCL (fc=0.5M KCl)
1.5ml of 1M CaCl2 (fc=0.15M CaCl2)
2.5ml of 1M MgCl2 (fc=0.25M MgCl2)
4.75 ml nanopure water
4.5 µl water
2 µl cut PCR (cleaned and in water)
2 µl cut plasmid (cleaned and in water)
1µl T4 ligation buffer
0.5µl T4 ligase
Ligate for 1-3 hrs at room temperature or for overnight at 4-16C in the fridge.
1µl-2µl of ligated plasmid
59µl of water
40µl of 5x KCM
100µl of competent cells Berglund:Making_Competent_Cells:_Various_Methods
- incubate on ice for 20 min
- heat shock for 90 sec at 42°C
- add 80µl SOC
- plate all of it on LB-amp
Cutting the plasmid and then gel purifying the plasmid helps to get rid of false positives (the insert you cut out will be lost and that is a good thing)
Cutting plasmid and insert longer is a good idea if you are not getting a high cutting efficiency (sometimes we do overnight cutting)
Ligation works more efficiently if you have a much higher ratio of insert to plasmid (10:1).
ligating longer can help with low efficiency
using more ligated product in the transformation may help if you are getting low numbers of colonies
Always run controls (uncut plasmid [expect lots of colonies], cut plasmid with no insert [expect no colonies], cut plasmid with insert but no ligase [expect no colonies])
The initial incubation on ice can be reduced to 2-5 min. However, if you are looking for high transfromation efficiency then you want to use the 20 min incubation.
If you are going to be using kanamycin on your plates, you need to rescue the cells adding soc and then shaking at 37° for 1 hr
If you are using ampicillin on your plates you do not need to rescue the cells
From K. Henscheid (8/8/2006):
I looked up info on transforming BIG plasmids, as the construct I've been struggling with is 11kb, and was wondering if I was having transformation issues. On www.promega.com/enotes/faqspeak/0107/fq0030.htm, they suggested a few ways to increase transformation efficiency:
o use high-efficiency competent cells [who doesn't??]
o use electroporation, which tends to have higher efficiency for all plasmid sizes.
o decrease antibiotic concentration, as large plasmids tend to have lower copy numbers.
I tried electroporation for my most recent cloning, and did a regular chemical transformation alongside. Since I had to do a recovery step after electroporation, I recovered the NovaBlues as well: 37 deg for 30 min. Both transformations appear to have worked well (by colony counts--my experimentals have more colonies than the negative control). So I'd suggest, if cloning doesn't appear to be working, do a recovery after transformation and before plating to increase transformation efficiency.
(It should also be noted that, in this round of cloning, I stacked the deck [I didn't know where my problem was]: There was no hint of a kit--every purification (post-PCR and post-digest/SAP) was one phenol/chloroform extraction, one chloroform extraction, and an ethanol precipitation with glycogen and washing with 75% EtOH. I quantitated insert and plasmid on the nanodrop and did the proper 3:1 insert:plasmid ratio with 100ng of plasmid. And I did the ligation overnight in the refrigerator. I also checked my ligations on an agarose gel for the heck of it. All things to try, if troubles are being had.)
Also useful: from the Promega T4 DNA ligase protocol, a quick way to figure out the 3:1 ratio is (kb insert * ng vector) / (kb vector) * 3 = ng insert.