IGEM:Harvard/2009/General Protocols

QIAprep Spin Miniprep Kit
This protocol is designed for the purification of up to 20 ug high-copy plasmid DNA from 1-5 ml overnight E. coli culture in LB medium.

Procedure

 * 1) Resuspend pelleted bacterial cells in 250uL Buffer P1 and transfer to a microcentrifuge tube.
 * 2) Add 250 uL Buffer P2 and mix thoroughly by inverting the tube 4-6 times.
 * 3) Add 350 uL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
 * 4) Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
 * 5) Apply the supernatant to the QIAprep spin column by pipetting
 * 6) Centrifuge for 30-60 s. Discard flow-through.
 * 7) Wash QIAprep spin column by adding 0.75 ml BUffer PE and centrifuging for 30-60s.
 * 8) Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
 * 9) To elute DNA, place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 uL Buffer EB to the center of each QIAprep, let stand for 1 min, and centrifuge for 1 min.

QIAGEN Plasmid Midi Protocol
This protocol is designed for preparation of up to 100 μg of high- or low-copy plasmid DNA using the QIAGEN Plasmid Midi Kit. Maximum recommended culture volumes

bacteria has been grown for a short time and the pellet is not enough. In that case use more bacteria (for e.g 50 ml)
 * 1) QIAGEN-tip 100
 * 2) High-copy plasmids 25ml
 * 3) Low-copy plasmids 100ml
 * 4) NOTE: For all practical purposes, we do not use more than 25 ml of bacteria unless the

Important notes before starting dissolve the SDS by warming to 37oC. spinning bacteria, spinning DNA etc.
 * 1) Make sure that RNase A has been added to Buffer P1.
 * 2) Check Buffer P2 for SDS precipitation due to low storage temperatures. If necessary,
 * 1) Pre-chill Buffer P3 to 4oC
 * 2) Figure out what tubes will you use at each steps. We have specific tubes assigned for

Procedure

 * 1) Centrifuge for 12 min to get bacterial pellet.
 * 2) Resuspend the bacterial pellet in 4 ml of Buffer P1 NOTE: Ensure that RNAse A has been added to Buffer P1. The bacteria should be resuspended completely by pipetting up and down until no cell clumps remain.
 * 3) Before adding Buffer P2, transfer into a centrifuge tube. Add 4 ml of Buffer P2, mix gently but thoroughly by inverting 4-6 times, and incubate at room temperature for 5 min. NOTE: The lysate should appear viscous.
 * 4) Add 4 ml of chilled Buffer P3, mix immediately but gently by inverting 4-6 times, and incubate on ice for 15 min. NOTE: Lysate should become less viscous.
 * 5) Centrifuge at 20,000 x g for 30 min at 4oC. Remove supernatant containing plasmid DNA promptly. NOTE: While waiting for step 4, finish step 5.
 * 6) Equilibrate a QIAGEN-tip 100 by applying 4 ml of Buffer QBT, and allow the column to empty by gravity flow.
 * 7) Apply the supernatant from step 4 to the QIAGEN-tip and allow it to enter the resin by gravity flow.
 * 8) Wash the QIAGEN-tip with 2 x 10 ml of Buffer QC.
 * 9) Elute DNA with 5 ml of Buffer QF.
 * 10) Precipitate DNA by adding 3.5 ml (0.7 volume) room-temperature isopropanol (also called 2-propanol) to the eluted DNA. Mix and centrifuge immediately at 15,000 x g for 30 min at 4oC. Carefully decant the supernatant. At this point you can sometimes see white pellet (DNA)
 * 11) Wash DNA pellet with 2 ml of room-temperature 70% ethanol, and centrifuge at 15,000 x g for 10 min. At this point you should/might see white pellet. Decant the ethanol very carefully.
 * 12) Add another 1ml of 70% ethanol to the tubes. Let the tube air dry, add some water, and then transfer the DNA pellet+ ethanol to an Eppendorf tube and spin for 2 minutes. Now the DNA pellet should be easily visible. Decant the 70% ethanol and spin again. Remove the traces of 70% ethanol as much as possible using pipette.
 * 13) Air-dry the pellet for 5-10 min, and redissolve the DNA in a suitable volume of buffer(TE). Usually we dissolve the DNA in 50 100 μl of TE.

Restriction Digest, using New England Biolabs (NEB) Enzymes
(You can add a little water or EB buffer to make this easy, without hurting the reaction) (Keep a few micoliters to run on a gel against the product, as a negative control) (See the enzyme(s) page in the NEB manual or website for the correct one) (BSA is recommended for some reactions (see manual), and will not hurt any) (Keep these on ice or in a freezer box, Always) (Also, keep the percentage of enzyme in the reaction below 5% by volume to avoid nonspecific cutting) (Overnight is ok)
 * Pick a volume where it will be easy to calculate the quantities of 10x and 100x buffers to add and write out a quick recipe (See samples).
 * Pipette the DNA to cut into a 1.5ml eppendorf tube.
 * Add water if you need (See example below)
 * Add 10X reaction buffer
 * Add 100X BSA if needed
 * Add restriction enzyme(s) last
 * Let the reaction run at the temperature recommended in the NEB manual for at least 2 hours.
 * Run a bit of the digest sample against the undigested control to confirm that the digstion worked.

Sample single digest (50 ul total volume)

 * 1) 30ul DNA
 * 2) 13.5ul Water
 * 3) 5ul 10X Buffer
 * 4) 0.5ul 100X BSA
 * 5) 1ul Restriction Enzyme

Sample double digest (50 ul total volume)
First, check the NEB manual double digest page for optimal conditions or whether it is recommended against for your enzymes
 * 1) 30ul DNA
 * 2) 12.5ul Water
 * 3) 5ul 10X Buffer (Check NEB double digest table - many not be what is used for the single digests
 * 4) 0.5ul 100X BSA (Add if it is required for either enzyme)
 * 5) 1ul Restriction Enzyme 1
 * 6) 1ul Restriction Enzyme 2

Filter paper gel extraction—Electroelution Dialysis DNA extraction protocol
Cut piece of filter paper into small square. Sandwich square and plastic. Do not let filter paper get wet. Used curved tweezers not flat because it will crumple the paper. Need to shove paper into gel into cut we made, in front of where the band we want to extract is. Grip it close to the bottom of it. The pastic is dialysis tubing. Also make a cut above the level of your band to extract so you can put a plastic tubing slice to block the unwanted bands from running into your desired one. You need to cut all the way through the gel and then shove the sandwich in flat to the bottom of the tray. Run the gel and within 15 minutes you will definitely have all of your DNA. If you run a lot of DNA you can see it in the gel without the light because it will be pinkish orange on the paper itself (b/c ethidium bromide). When you put the filter paper in the column, put the DNA side down so the papr does not block the DNA going down. Orient the paper in the tube depending on where the filter paper is relative to the membrane. You can spin it down, rehydrate it, and spin it down again. You should add about 50 uL of water, max 100 uL. Then you will take filter paper and elute into a UltraFree-MC filter column, and then take the eluant and put that onto a PCR purification column. With E-gels, they are 9 dollars each so load as many samples as you can.

Gene assembly protocol
Design oligos of ~36-45 nt long spanning your entire gene/sequence of interest plus any necessary flanking sequences for both strands that anneal with 18-22 bp overlaps as follows:



1- KINASE REACTION

Resuspend oligos to a concentration of 100 uM.

Mix together equal amounts of each oligonucleotide.

Prepare kinase reaction as follows:

20 uL H2O

6 uL oligomix (100 uM)

4 uL 10x T4 PNK buffer

4 uL 0.1 M DTT

4 uL 10 mM ATP

2 uL T4 PNK enzyme

--

40 uL total

Incubate 20 min @ 37C

Heat inactivate 2 min @ 94C

Cool 0.1C/sec to 70C

2- LIGATION

Pre-warm the following to 70C and add to kinase reaction:

50 uL H2O

10 uL 10x DNA ligase buffer

---

100 uL total

Incubate 60 min @ 70C

Cool 0.1C/sec to 25C

take out 5uL as "before ligation" sample for check gel.

Add 2 uL T4 DNA ligase

Incubate at room temperature for 2 hours

3- PCR

Mix together the following:

39 uL H2O

5 uL 10x Thermopol Buffer

1 uL dNTPs

1 uL Forward Primer

1 uL Reverse Primer

2 uL Ligation reaction

1 uL Vent DNA polymerase

50 uL total

Run PCR program:

1) 5 min @ 95C

2) 30 sec @ 95C

3) 30 sec @ 50C

4) 10 sec @ 72C

5) Repeat steps 2-4 30 times

6) 4C forever

Qiagen purify the PCR product, nanodrop to determine DNA concentration, and run a restriction digest to prepare the product for ligation.

Run a check gel with the pre-ligation oligo mix, post ligation reaction mix, and PCR product.

To transform chemically competent cells

 * 1) Start thawing the competent cells on wet crushed ice.
 * 2) Chill labeled 2 ml conical bottom tubes on wet ice. Add 1 µL of DNA in TE and 50 µL of thawed TOP10 competent cells to the tubes. In our experience, these volumes have the best transformation efficiency. The 2 ml tubes allow better liquid movement during incubation. Extra eluted DNA may be held at least several weeks frozen or at refrigerator temperature.
 * 3) Hold the DNA and competent cells on ice for 30 minutes. This improves transformation efficiency by a significant amount.
 * 4) Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. A water bath is important to improve heat transfer to the cells.
 * 5) Incubate the cells on ice for 2 minutes.
 * 6) Add 200 μl of SOC broth (check that this broth is not turbid, which would indicate previous contamination and bacterial growth). This broth should contain no antibiotics.
 * 7) Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. We have found that growth for 2 hours helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin.
 * 8) Label an LB agar plate containing the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance. Plate 250 µl of the incubated cell culture on the plate.
 * 9) Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics, especially ampicillin, start to break down and un-transformed cells will begin to grow.