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.

Restriction Digest, using New England Biolabs (NEB) Enzymes

• 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).

(You can add a little water or EB buffer to make this easy, without hurting the reaction)

• Pipette the DNA to cut into a 1.5ml eppendorf tube.

(Keep a few micoliters to run on a gel against the product, as a negative control)

• Add water if you need (See example below)
• Add 10X reaction buffer

(See the enzyme(s) page in the NEB manual or website for the correct one)

• Add 100X BSA if needed

(BSA is recommended for some reactions (see manual), and will not hurt any)

• Add restriction enzyme(s) last

(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)

• Let the reaction run at the temperature recommended in the NEB manual for at least 2 hours.

(Overnight is ok)

• 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.