Protocol for Stbl3 competent cells

[CB]

Does anyone have a protocol for making Stbl3 competent cells? We buy them from Invitrogen and use them a lot so it is getting expensive. Therefore I would like to make our own stock in lab. Please let me know if you are willing to share your protocol. 

Thanks!

[Mark]

This works great for us even when transforming ligation reactions. Starting from 1L gives you a lot.

Preparation:

·         Use glycerol stock or stock of existing competent Stbl2 to streak cells for single colonies on LB plate without antibiotics and grow over night in bacterial incubator (37°C). Store plate at 4°C. Plate should be good for at least 2 weeks.

·         Buffers:

TFB1 (500ml)	TFB2 (500 ml)
KAc                        1.5g	MOPS                   1g
MnCl2:4H2O        7.85 g (5g without H2O)
KCl                          3.75 g	KCl                          0.375g
CaCl2 (2.5M)       3.2ml	CaCl2 (2.5M)       15.2ml
Glycerol (100%)                67 ml	Glycerol               67ml
Adjust pH to 5.8 with 1M HCl (few drops only!)	Adjust pH to 7.0 with KOH.

 

o   Filter sterilize the buffers

o   Make buffers fresh every time (not sure whether this is necessary).

 

Protocol:

Evening:

·         Inoculate 20-30 ml LB (without antibiotics) from plate (pick single colony).

·         Grow over night in bacterial shaker (37°C)

Next morning:

·         inoculate 1L of LB with starter-culture (1:200 to 1:66). [keep 1 ml of the same batch of LB as a blank for spec.]

·         Incubate in bacterial shaker until OD(600) is at ~0.5. [start measuring ~2hrs after inoculation of culture, assume population doubling every 20-30 min. E.g., if first value is 0.25, measure again 20-30 min later. Bacteria should not be at an OD higher than 0.5-0.6 to make competent cells.]

·         Pour culture into clean centrifuge tubes (e.g. tubes that are reserved to make competent bacteria, i.e. are free of plasmid contamination, or disposables) and incubate on ice for 10 min.

Bacteria should be kept as cold as possible from now on. Steps can be performed in cold-room.

·         Pellet at 4°C (3200rpm, 10min), resuspend each pellet (from 500ml culture) in 10ml TFB1. Bring volume up to 200 ml with TFB1.

·         Incubate on ice for 10 min.

·         Pellet at 4°C (3200rpm, 10min), resuspend each pellet in 30-50ml TFB2.

·         Incubate 10 min on ice.

Aliquot into pre-cooled 1.5 ml or 0.5ml tubes (on ice or in cold-room) and snap-freeze.

[Mark]

PS/ Protocol says Stbl2 but we have been using this for Stbl3 as well.

[CB]

Thanks for sharing!

[Phil Abbosh]

Hi Mark, thanks for sharing your protocol. when transforming, do you just mix your bugs and plasmid and streak or do you have to grow out in SOC? Also, this is for heat shock transformation I assume, not electroporation, correct?

phil

[Mark]

Hi Phil,

This is for heat-shock.

It depends on what I'm doing for transformation. If it's just direct transformation of plasmid (ex. if you're getting a plasmid from Addgene), I just spread 5 uL (of a 50 uL reaction) onto a LB-plate. If it's ligation, I recover in 500 uL LB (or SOC if you want) at 37C for 15-45' then plate everything (length of incubation depends on how difficult your ligation is).

[Trisiani Affandi]

Hi Mark,

Thanks for sharing your protocol. Quick question, this protocol doesn't use rubidium? Is there a reason why? Have you tried making Stbl2 competent cells just using CaCl2 competent cell prep?

Thanks!

Trisiani

[Mark]

Hi Trisiani,

I believe this protocol was from the Molecular Cloning: A Laboratory Manual so this has already been established. I don't really know anyone who uses rubidium to be honest. Since undergrad, all the protocols I've seen always use Ca/Mn salts. Looking at this abstract also suggests rubidium is the least effective.

I have never used only CaCl2 with Stbl2. I think I used a similar one with the Ca/Mn (no K) in grad school. Worked for me but I didn't really do any side-by-side comparisons.

Hope this helps.

Mark

[Alison Knowles]

Hi Mark,

My lab is having some trouble with making STBL3 competent cells and I was wondering if you could give some advice...

I think where we go wrong is during the transformation. Do you do anything different from typical transformation protocols for the STBL3 cells? If so, could you upload that protocol? The cells grow on antibiotic plates after we made them competent, but with each time I test the transformation efficiency it seems to get lower and lower... I'm wondering of they have a short shelf life or if simply we are doing something wrong. Thanks!

-Ali

[Bowen Zhou]

Hi Ali,

In our lab's experience, the most important parts of making Stbl3's are to ensure that the cells are on ice the entire time after the step which specifies it and that the the bacterial flasks are completely washed (5 times for us) to get rid of detergent. Shelf life at -80 ºC should be quite long.

Bowen

[A.J. Boender]

Hee,

I can really recommend the mix and go kit from zymogen. https://www.zymoresearch.com/products/mix-and-go-e-coli-transformation-kit

It was recommended to me by Addgene and it works really well. You just have to buy the Stbl3's from Invitrogen once and then you can make a glycerol stock from them that lasts pretty much forever.

Then, if you made the stbl3's with this kit, you don't have to heat shock or anything, just mix the plasmid with the cells, plate them and be happy with the result the next day!

The kit allows for the generation of a LOT of aliquots, so it is def. much cheaper.

Best,

Arjen

Op vrijdag 1 juli 2016 08:59:18 UTC-4 schreef CB:

[Haajira Bv]

I made competent cell preparation with this protocol for stbl3 and I Compared this efficiency with DH5-alpha, and DH10-beta (comp. cell by cacl3 method). but the results I m getting efficiency of stbl3 is lesser than those two strains while transferring the lentiviral 3rd generation plasmid (pljm1). Can anyone explain where will be the issue?

[Garrett Montgomery]

making competent cells can be tricky.  Most people dont get the same efficiency as you will with store bought cells.  That being said, its not impossible.   The best advice I've ever been given which made drastic improvements in the efficiency was to grow everything cold.  You can grow your starter culture at 37C (although best to do at room temp) but its crucial  to grow your culture for making competent cells at 18-22C.  Following this advice brought the efficiency of my cells up to store bought quality. Once grown to an OD600 value somewhere between 0.4-0.6, it is time to harvest.  I prefer to use the mix and go kit from zymo. My protocol is as follows 

1- streak plate from store bought tube of cells 

2- incubate at 30C overnight.

3- pick colony for starter culture and grow at room temp overnight.

4- inoculate 100mls of ZymoBroth the following morning.

5- grow in a shaker at 18-22C

6- at 2 hours begin checking the OD read every thirty mins until you reach 0.4-0.6 value.

7- remove from shaker, enter into the cold room (or be like me and put your incubator in the cold room to start) and place the cells on ice for 10 mins 

from there forward I just follow the steps of the zymo mix and go kit.  I make sure to carry out the entire protocol in the cold room and then snap freeze the e.coli in a aluminum reaction block on dry ice.