Team:TPHS SanDiego/Protocol

From 2013hs.igem.org

Preparing Agar Plates with Ampicillin
Ampicillin is added to agar plates to test the success of the plasmid transformation. (Ampicillin resistant genes are added to the E.coli).
CPEC
Cloning method for assembling PCR fragments together. I typically set my overlapping regions to a Tm of ~60-65C and my PCR annealing region to a Tm of ~57-60C.
OLD: PCR purify each piece , then setup a PCR reaction with these requirements:
NEW: Gel Purify each piece , then setup a PCR reaction with these requirements:
about ~300ng of vector
about ~200ng of insert
1:1 molar ratio of pieces
PCR mix (amounts in microliters)
a. 10.0 HF buffer
b. 1.0 dNTPS
c. 0.5 Phusion (polymerase)
d. PCR fragments
g. fill up to 50 with qH20

CPEC protocol is:
98C 30sect
30 cycles of:
98C 10sec
55C 30sec
72C 20sec/kb
72C 5mins
Before running CPEC protocol, save 10 uL on ice(freezer) as a control.
After CPEC is done, run 10 microliters of the CPEC reaction with the control in the next lane. Check that you can see your assembled reaction on the gel, or that your inserts are less visible than the control (they assembled). If so, then transform 10 microliters into regular competent cells.
Note: In some situations it is difficult to fulfill the first 2 requirements (200 ng insert & 300 ng vector). In those cases I would just make sure there is enough vector, and add enough insert at a insert:vector of (1:1-3:1)
Note2: I've found recently that Gel Purifying the vectors gives much higher efficiency and much cleaner results when running the CPEC on a gel. This seems to reduces smears in some CPEC reactions or strange sized pieces(also what they do in paper). I would recommend running PCRs of 50-100 microliters and then gel extracting directly after the PCR instead of gel checking & PCR purifying.



Wobble PCR
EIPCR (enzymatic inverse PCR)
Normal ligation assembly (3A assembly)


Mini-preps
1) Resuspend pelleted bacterial cells in 250 µl (320 µL) Resuspension Buffer and transfer to a microcentrifuge tube.
2) Add 250 µl (320 µL) Lysis Buffer and mix by gentle inversion the tube 4–6 times to mix. Do not vortex, as this will result in shearing of genomic DNA
3) Add 350 µl (450 µL) Neutralization Buffer and invert the tube immediately but gently 4–6 times.
4) Centrifuge for 10 minutes at 13,000 rpm in a table-top microcentrifuge.
5) Transfer the supernatant in a clean Eppendorf tube.
6) Add 0.6 volumes of isopropanol.
7) Centrifuge 10 minutes at maximum speed in a table top centrifuge
8) Wash with 70% ethanol.
9) Air dry and resuspend in 10 mM Tris pH = 7.4 (or ddH 2O)


Transformations
1.Thaw competent cells on ice.
2.Chill approximately 5 ng (2 μl) of the ligation mixture in a 1.5 ml microcentrifuge tube.
3.Add 50 µl of competent cells to the DNA. Mix gently by pipetting up and down or flicking the tube 4–5 times to mix the cells and DNA. Do not vortex.
4.Place the mixture on ice for 30 minutes. Do not mix.
5.Heat shock at 42°C for 30 seconds*. Do not mix.
6.Add 950 µl of room temperature media* to the tube.
7.Place tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8.Warm selection plates to 37°C.
9.Spread 50–100 µl of the cells and ligation mixture onto the plates.
10.Incubate overnight at 37°C
* Please note: For the duration and temperature of the heat shock step as well as for the media to be used during the recovery period, please follow the recommendations provided by the competent cells’ manufacturer.


Ligations
1.Combine 50 ng of vector with a 3-fold molar excess of insert. Adjust volume to 10 μl with dH2O.
2.Add 10 μl of 2X Quick Ligation Buffer and mix.
3.Add 1μl of Quick T4 DNA Ligase and mix thoroughly.
4.Centrifuge briefly and incubate at room temperature (25°C) for 5 minutes.
5.Chill on ice, then transform or store at -20°C.
6.Do not heat inactivate. Heat inactivation dramatically reduces transformation efficiency.


Gel Digests
During digestion, DNA is cut at specific sequences, and sticky ends are created. Sticky ends are created for future ligation of DNA.

There are two types of Digests we do with restriction enzymes:
(1) screening/diagnostic of colonies
(2) digesting PCRs/plasmids for gel extractions & ligations.

(1) For diagnostic gels, we use between 50-200ng of DNA and digest for 1/2-1 hour before running on a gel. This is just to check if our DNA has the correct fragments
(2) For gel extractions, we use between 1500-3000ng of DNA and digest for 2-3 hours before running on a gel. The gel extraction procedure has low yield thus a lot is needed to start with.

Type (1) Diagnostic Gels
MIX: Typically 1.0-4.0 microliters DNA, 1.0 of each 10x buffer (check chart), 0.25-0.5 of each enzyme, fill up to 10.0 microliters total with water.
Leave at 37 incubator for 1/2-1 hour
Make gel with thin comb
By the time gel solidifies, probably ready to run gel
Typical Mix is
2.0 DNA
1.0 Buf 2
1.0 BSA
0.5 KpnI
5.5 H20
10.0 TOTAL

Type (2) Gel Extractions
Digest desired amount of DNA (will probably be around 30-60 microliters) for 2-3 hours at 37.
Make wide gel comb
Run gel till bands are well separated and cut out gel piece, trying to minimize amount of agarose
Typical Mix
46.0 DNA
6.0 Buf 2
6.0 BSA
1.0 KpnI
1.0 MluI
60.0 TOTAL
Note: After creating mix, vortex briefly, then spin down briefly to ensure consistency.
Note: Addition of BSA does not affect digests so if 1 enzyme requires it, just add it.


Normal PCRs

To amplify fragments of DNA
New PCR Mix: This mix is a little better and allows you to keep primers undiluted and gives more product for CPEC's

Phusion: Keep in -20 holder, add to mix last
1. PCR mix (amounts in microlters.
a. 20.0 HF buffer
b. 2.0 dNTPS
c. 0.5 Phusion (polymerase)
d. 0.5 primer –S (undiluted, 100uM)
e. 0.5 primer –AS (undiluted, 100uM)
f. ~1.0-2.0 template DNA (need 20-40ng of DNA)
i) If template is at X concentration, make a dilution to get it to 20-40ng/uL.
g. 75.0 qH20


Old PCR Mix: PCR primers should be at concentration 10.0 uM

Before Making Mix Below: Prepare primers
We buy primers at 100.0 uM (pmol/microliter) concentration. For the mix below they need to be at 10.0 uM (10x dilution).
Label a new 0.6 mL microcentrifuge tube with the primer name.
Add 90 microliters buffer EB
Add 10.0 microliters of primer
Vortex briefly


For Phusion: Keep in -20 holder, add to mix last
1. PCR mix (amounts in microlters.
a. 10.0 HF buffer
b. 1.0 dNTPS
c. 0.5 Phusion (polymerase)
d. 2.5 primer –S
e. 2.5 primer –AS
f. 0.5-1.0 template DNA (need 10-20ng of DNA)
i) If template is at X concentration, make a dilution to get it to 10-20ng/uL.
g. 33.0 qH20


Common protocol
98ºC 2mins, [initialization]
32x
98ºC 10 sec [denaturation]
50-65ºC 15-30 sec [annealing]
72ºC for 15-20sec/kb [extension]
72ºC for 40sec/kb.

Gel-extractions
PCR-purifications
Running electrophoresis gels
Comp cells

0. Start an O/N culture of your strain in 1-5mL LB media.
1. 1/100x dilution of overnight culture in 25 mL LB (makes 25 competent cells). Grow up for ~2-3 hours(depending on strain), OD needs to be between 0.4-0.6 for significant competency.
2. Spin down cells at 8000 for 5 min. Resuspend in 2.5 mL TSS solution(1/10x original). Aliquot to prechilled microcentrifuge tubes (prechill at -20C). Freeze at -80C for storage (retain competency for about ~6 months)


Plating
Overnight cultures (liquid cultures)


Denote when the UCSD services had to be used – sequencing, designing primers, data managing techniques, ordering primers, thermocycler