The following protocols go over how to design the DNA sequence for your peptide library and also how to clone it into the pre-cut vector arms that come with the T7 select systems. The general approach is to (1) design/create blunt ended, dsDNA segments with restriction sites (RS) flanking the peptide sequence, (2) purify this DNA, (3) digest it with restriction enzymes (RE) to create sticky ends, and (4) ligate it into the vector arms.
(1) To create the dsDNA insert encoding your peptide, I recommend ordering one long oligo (forward primer) containing the entire RS1-insert-RS2 sequence and one short reverse primer that can anneal to the end of the forward primer and extend to create the desired dsDNA. If you are ligating into the pre-cut vector arms that come with the system, then RS1 should be the EcoRI restriction site and RS2 should be the HindIII restriction site. You can use the template below for designing your forward primer:
5'- GCATGGAATTCG - peptide sequence - TAAGCTTGGACTGCT -3'
The underlined regions are the EcoRI and HindIII restriction sites, respectively. The base-pairs outside of these sites just provide buffer space for better RE binding and (at the 3' end) reverse primer attachment. Also, note that the TAA codon following the peptide sequence is the stop codon. So your peptide will terminate after the insert using this design.
For creating random peptide libraries, you'll want to put a degenerate sequence in the "peptide sequence" region. A popular choice is repeats of the codon NNK. N is an equal mixture of A,G,C,T and K is an equal mixture of G,T. The NNK sequence codes for a 32 codon scheme: 1 stop codon, 1 codon for each of 12 amino acids, 2 codons for each of 5 amino acids, and 3 codons for each of 3 amino acids.
You can use the following design for the reverse primer:
5'- AGCAGTCCAAGCTTA -3'
The Tm for this forward primer/reverse primer annealing is around 49 degrees C.
Someone in the lab who has used T7 systems might already have this reverse primer.
Once you have your forward and reverse primer, you can create the bunt end, dsDNA insert as follows:
1. in a 1.5 mL eppindorf tube mix:
- 400 pmol forward primer
- 400 pmol reverse primer
- 40 uL of 5x phusion buffer
- 152 uL of ddH2O (to a total volume of 200 uL)
2. Incubate at 70 degrees for 15 min (I used the eppindorf thermomixer)
3. Slowly drop temperature to rt over 30 minutes (I just slowly decreased temp of the incubator).
4. add 4 uL of 10 mM dNTP (to 200 uM), then add 4 uL Phusion (to 2 U/50 uL).
5. incubate for 30 min at 37 degrees
6. incubate for 5-10 min at 72 degrees.
7. store at -20
(2) Now, we need to purify the DNA from the Phusion in order to digest it. To do this, run 50 uL of the solution on a gel, slice out the band with a razor, and place the gel slice into a pre-weighed eppindorf tube. You can weigh the tube again to get the weight of the gel slice. Then you can calculate the volume of the slice using the density of a 1% gel, which is similar to that of water (1 mg/uL). If the dsDNA segment is greater than 70 bp, you can extract it from the gel with the QIAGEN extraction kit. Otherwise, use the following protocol:
1. Mash up the gel slice in the bottom of the eppindorf tube (I did this by pressing it down with the tip of a sterile plate spreading stick).
2. Add 500 uL TE buffer to the tube. And leave it shaking overnight (i used 750 rpm in the eppindorf thermomixer at room temp). The next day, the concentration of DNA in gel/TE should hopefully have reached an equilibrium.
3. Extract the TE by spinning down the gel in the eppindorf tube for 30 minutes at maximum rpm and pipette out 450 - 500 uL of the solution into a separate eppindorf tube (use nanodrop to measure concentration at this point).
4. Run ethanol precipitation (as follows):
4.1 add1/10 volume sodium acetate to the solution
4.2 add 2 volumes of absolute ethanol (pre-chilled at -20).
4.3 let incubate for 1-2 hrs at -80 (the smaller the size/concentration of DNA, the better it is to leave it incubating longer.. i used 2 hrs and then left it at 4 degrees for another 10 min)
4.4 centrifuge at max rpm for 30 min at 4 degrees (I moved the microfuge into the cold room for this).
4.5 aspirate the ethanol from the DNA pellet (get as much as possible without disrupting the pellet).
4.6 wash with 1 mL 70% ethanol (pre-chilled at -20). To do this part, slowly pipette the ethanol over the pellet, then slowly invert the tube back and forth. Try not to break up the pellet.
4.7 Centrifuge the pellet back down at max rpm for 2 min.
4.8 remove the ethanol from the pellet (I did this by aspirating most of it with a pipette and then using a speed vac (unheated) the evaporate the remaining).
4.9 resuspend the DNA pellet in water to desired concentration (I used 30 uL).
5. store at -20
(3) You can do a double digest of your purified dsDNA with EcoRI-HF and HindIII (both from NEB) in NEBuffer2. DO NOT use regular EcoRI and HindIII because of star activity.
Here is the protocol I used:
1. Mix the following in a 1.5 mL eppindorf tube
- .1uL EcoRI-HF (100000 U/uL) to 10 U
- .5uL HindIII (20000 U/uL) to 10 U
- 1ug DNA
- 5 uL of 10x NEBuffer2
- fill to 50 uL with ddH2O
2. incubate at 37 degrees C for 2-2.5 hrs.
3. Purify the digest according to the previous protocol (2). Aim for a final concentration of around .06 pmol/uL
4. store at -20.
(4) Ligate the purified insert (digest) into the vector arms with the following protocol:
1. mix the following in a PCR tube:
- .06 pmol of insert (1 uL of .06 pmol/uL insert)
- 1 uL of T7 select vector arms
- .5 uL of 10x Ligase buffer
- .15 uL of T4 Ligase (400000 U/mL from NEB). This is roughly 60 Cohesive End Units.
- fill to 5 uL with ddH2O
2. keep at 16 degrees C for 16 hours.