TITLE: Intein-assisted Bisection Mapping (IBM)
AUTHORS: Trevor Ho, Baojun Wang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Split inteins are powerful tools for seamless ligation of synthetic split proteins. Yet, their use remains limited because the already intricate split site identification problem is often complicated by the requirement of extein junction sequences. To address this, we augmented a mini-Mu transposon-based screening approach and devised the intein-assisted bisection mapping (IBM) method. IBM robustly revealed clusters of split sites on four proteins, converting them into AND or NAND logic gates. In this protocol we provide details in executing the IBM method.</div></div>

[STEPS]
?. [Introduction]
IBM starts with a transposition reaction: a staging vector carries a 5’ and 3’ trimmed gene of interest (GOI) with the internal BsaI, BbsI and SapI sites removed. The staging plasmid is mixed in vitro with the MuA transposase and the mini-Mu transposon, which generates the initial insertion library. After in vivo amplification (transformation into E. coli and overnight growth), the insertion library is cut by BsaI and resolved on a DNA agarose gel. The band with the correct size corresponding to GOI with the insertion is purified and ligated into a linearized, protein expression vector with BsaI-generated overhangs. This produces the Open Reading Frame (ORF) insertion library. The ORF insertion library is again amplified in vivo and then an aliquot is mixed with a DNA fragment carrying the split gp41-1 intein intein in a Golden Gate reaction, using the restriction enzymes BbsI and SapI. The DNA fragment contains a different selection marker (cat) to facilitate selection of the library with the transposon replaced. In the end, the final library is screened for individual strains that showed proper reconstitution of protein function, i.e. resistance against ampicillin upon expression of both the N- and C-lobes (AND logic gate behavior). Those strains were then subjected to Sanger sequencing the C-terminal joint to map the split sites. Identical split sites were then deduplicated and consolidated with activity data to generate the intein-bisection map.This protocol describes the main procedures of transposition, size selection, substitution, and mapping. Molecular cloning to generate the starting DNA constructs are not covered. However, this protocol will start with describing the necessary treatment on those constructs once they are available.The most important objective in this protocol is to obtain a final library with large enough "library complexity, or diversity", in other words, ensuring that during construction and the final phase, the libraries contain enough random events so one can be confident that most possible splits are covered, and that a function split site for intein can be screened out.
?. [Preparation of DNA materials]
Grow and harvest 10 mL of bacterial culture of the protein expression vector.Perform a mini-prep the next day.
?. [Preparation of DNA materials]
Prepare linearized protein expression vector
?. [Preparation of DNA materials]
Grow and harvest  2 *  of the protein expression vector.Perform a mini-prep the next day.Pool the extracted DNA into one tube.
[bacterial culture ]
?. [Preparation of DNA materials]
Digest   from miniprep using.Simultaneously treat with  .  ABC1Number of reactions15210X Cutsmart Buffer2103BsaI-HFv2 (20 U/μL)154FastAP (1 U/μL)155DNA (concentration: 89 ng/μL)11.2566water4.824Run reaction at   for at least  .
[ plasmid of protein expression vector]
ABC1Number of reactions15210X Cutsmart Buffer2103BsaI-HFv2 (20 U/μL)154FastAP (1 U/μL)155DNA (concentration: 89 ng/μL)11.2566water4.824
37 °C
?. [Preparation of DNA materials]
Prepare the staging plasmid
?. [Preparation of DNA materials]
Resolved digested DNA on 0.5 - 1% agarose gel, depending on the size of the vector. Load  per lane.Excise the band corresponding to the linearized vector.
[digested DNA]
?. [Preparation of DNA materials]
Purify the DNA.Load at most 2 gel slices (1 slice per lane on gel) per column.Elute DNA from each column using  .Pool eluted DNA together and quantify the concentration.
[nuclease free water]
?. [Preparation of DNA materials]
Prepare the linearized mini-Mu transposon
?. [Preparation of DNA materials]
Grow and harvest  for the plasmid carrying the MuA-transposon (plasmid ID = IBMc090).Perform a midi-prep the next day.
[bacterial culture]
?. [Preparation of DNA materials]
Digest from miniprep usingand  .  ABC11X20210X Cutsmart Buffer2403DNA (1000 ng/μL)1204BglII (10 U/μL)1205BsaI-HFv2 (20 U/μL)1206water15300Run reaction at  , for at least  , or  if time is permitted.
[mini-Mu containing plasmid]
ABC11X20210X Cutsmart Buffer2403DNA (1000 ng/μL)1204BglII (10 U/μL)1205BsaI-HFv2 (20 U/μL)1206water15300
37 °C
?. [Preparation of DNA materials]
Resolved digested DNA on 1% agarose gel. Load   per lane.Excise the band with size of 1667 bp, which corresponds to the linear transposon.
[digested DNA]
?. [Preparation of DNA materials]
Purify the DNA.Load at most 2 gel slices (1 slice per lane on gel) per column.Elute DNA from each column using Pool eluted DNA together and quantify the concentration.
[nuclease free water]
It is important to obtain > 80 ng/μL of purified linear transposon for downstream processes.If your purification efficiency is too low, try using a to concentrate the DNA.
?. [Preparation of DNA materials]
Grow and harvest  for the staging plasmid.Perform a mini-prep the next day.
[bacterial culture]
?. [Transposition using an engineered mini-Mu transposon]
Prepare MuA transposaseThe MuA transposase should be stocked at  at high concentrations to maximize shelf-life.Dilute the concentrated MuA transposase to 220 ng/μL right before setting up the transposition reaction.Using leftover MuA transposase from previous experiments, even if kept at  , could lead to a much reduced transposition efficiency.
-80 °C
-20 °C
?. [Transposition using an engineered mini-Mu transposon]
ABC1Number of reactions1622X Mix12.57532.5 M NaCl + 0.25 M MgCl21641.25% Triton X-100165MuA Transposase  (220 ng/μl)3186Transposon (69.6 ng/μl)4.325.87Vector  (158 ng/μl)1.37.88H2O1.911.4Aliquot all mixtures into 2 *  in 200 μl PCR tubes. Run reactions on a thermocycler for1.    for 2.   for
[transposition reaction]
ABC1Number of reactions1622X Mix12.57532.5 M NaCl + 0.25 M MgCl21641.25% Triton X-100165MuA Transposase  (220 ng/μl)3186Transposon (69.6 ng/μl)4.325.87Vector  (158 ng/μl)1.37.88H2O1.911.4
[reactions]
30 °C
80 °C
?. [Transposition using an engineered mini-Mu transposon]
Pooled the reaction mixture together and purify the DNA library.Use a single column and elute all DNA in .It is not necessary to quantify the DNA concentration.
[nuclease free water]
?. [Electroporation of DNA library into E. coli]
Electroporate the DNA library into .
?. [Electroporation of DNA library into E. coli]
Thaw and then mix   with the eluted DNA library in a pre-chilled microcentrifuge tube. Place the tube on ice for
[electrocompetent cells ]
?. [Electroporation of DNA library into E. coli]
Aliquot   into a Ensure that the mixture is loaded in the gap. Place the cuvette on ice for
[DNA-competent cell mixture]
?. [Electroporation of DNA library into E. coli]
Take out the warmed up Stable Outgrowth Medium.Wipe down any traces of water on the cuvettes. Carry out electroporation using Use protocol EC1. Between each cuvette, jot down the time constants for electroporation event. The value should be between 4 to 5.5 ms. Even if the value is out-of-range, keep going.Immediate after electroporation, add into the electroporation cuvette. Gently suspend cells by mixing up and down for 1-2 times. Transfer all suspended cells in medium into the microcentrifuge or universal tube where the medium was warmed. For samples of the same library, pool the suspended cells together.It may be helpful to do this aseptically, but this is not strictly required. Speed is more important.
[Stable Outgrowth Medium]
?. [Electroporation of DNA library into E. coli]
Aliquot 4 *  of (come with the electrocompetent cells) into microcentrifuge tubes or 30 mL universal tubes / test tubes and warm up the medium in tubes at  for at least.
1 mL
37 °C
?. [Electroporation of DNA library into E. coli]
After finishing the electroporation steps for all samples, incubate the tubes at   for recovery.
37 33
?. [Electroporation of DNA library into E. coli]
Retrieve the recovered cells.For each library, there should be ~ 4 mL of recovered cells. Take out  and dilute it into  . Serially dilute it again 10-fold for 5 times. Label the tubes from A (lowest dilution) to E (highest dilution).Spread   onto 90 mm LB agar + KAN + CHL plates labelled A to E. This is to quantify the "library complexity".
[recovered cells]
[0.85% saline]
[diluted cells]
?. [Kits]
We found good quality of DNA from each step is essential for the success of the protocol.We use the following kits:  for miniprep for midiprepfor gel and all other DNA purifications and concentrating steps.
?. [Electroporation of DNA library into E. coli]
For the rest of the recovered cells, spread onto 4 * 145 mm LB agar + KAN + CHL plates, each using ~ . Allow the plates to dry before inverting them upside down.Incubate all plates overnight at
[recovered cells]
37 °C
?. [Electroporation of DNA library into E. coli]
Retrieve the plates and determine library complexity.For each library, tally the colony counts from plates A - E and create a table like below:If the library fold coverage is > 100. Proceed. Otherwise, discard the library.
ABCD1Plate# of coloniesdilution factorInitial number of transformants2ATMTC400-3BTMTC4000-4CTMTC40000-5D48400000192000006E4400000016000000 For n bp there is n-1 possibilities of insertion, and the transposon can insert in both forward and reverse orientations.Total number of colonies (aka library complexity) was 1.92 * 107.Size of staging plasmid is 2859 bp.Therefore, library fold coverage was 1.92 * 107/ ((2859 - 1) * 2) = 3359 fold
?. [Cell scraping and midiprep]
Scrap down all cells from the 4 * 145 mm plates using a L-shape spreader and 0.85% saline. For each plate, add  to each plate, scrape down cells and transfer cells in saline to a 50 mL centrifuge tube. Repeat this once.
[0.85% saline]
?. [Cell scraping and midiprep]
Centrifuge all tubes at  , remove most of the supernatant until the volume is , then resuspend the cell pellets completely. Pool all resuspended cells into one of the four tubes. Repeat centrifugation with a balance.
Centrifuge: 5000 33, 10 min
10 mL
?. [Cell scraping and midiprep]
Remove supernatant again until total volume is about . At this the suspension is very dense and probably a bit viscous. Take out  and save it as a glycerol stock.The glycerol stock can be re-propagated and will save huge amount of time if you need to reuse the library.
5 mL
[resuspended cells]
?. [Size selection on initial insertion library to generate ORF insertion library]
Digest  using   ABC1Number of reactions110210X CS Buffer2203BsaI-HFv2 (20U/μL)1104DNA (769 ng/μL)1.3135water15.7157Run reaction at  , for at least  , or  if time is permitted.
[ extracted, initial insertion library]
ABC1Number of reactions110210X CS Buffer2203BsaI-HFv2 (20U/μL)1104DNA (769 ng/μL)1.3135water15.7157
37 °C
?. [Cell scraping and midiprep]
Repeat the centrifuge again and this time, discard all supernatant. Perform a midiprep to extract the initial insertion library.
?. [Size selection on initial insertion library to generate ORF insertion library]
Resolved digested DNA on 0.5 - 1% agarose gel. The gel percentage will depend on the expected sizes.Load  per lane.Critical: Run the gel at slow speed, e.g. 75 V for 90 mins. Resolution between bands is extremely important here, because the band corresponding to the trimmed CDS inserted with the transposon could be faint and close to other bands.Excise the band which corresponds to the trimmed CDS inserted with the linear transposon.Example in calculating the theoretical size of the trimmed CDS inserted with the transposonThe staging plasmid for β-lactamse is 2859 bp. When digested by BsaI, it yields 771 bp (trimmed CDS) and 2088 bp (backbone).Therefore, after transposition:, each receives + 1667 + 5 bpExpected sizes of digested library are therefore 771, 2088, 2443, 3760 bpThe 2443 bp band is the band containing the trimmed CDS inserted with transposon
[digested DNA]
?. [Size selection on initial insertion library to generate ORF insertion library]
Purify the DNA.Load at most 2 gel slices (1 slice per lane on gel) per column.Elute DNA from each column using  .Pool eluted DNA together and quantify the concentration.
[nuclease free water]
?. [Size selection on initial insertion library to generate ORF insertion library]
Set up ligation of purified DNA to protein expression vector using the Use 1:2 insert:vector ratio to increase the chance that each insert will receive a vector.At this stage, the ligation volume is typically , but this can vary.  AB1Number of reactions1210X T4 DNA Ligase Buffer103Vector (119 ng/μL)94Insert (6 ng/μL)465T4 DNA Ligase106water25Run the ligation at   for
100 µl
AB1Number of reactions1210X T4 DNA Ligase Buffer103Vector (119 ng/μL)94Insert (6 ng/μL)465T4 DNA Ligase106water25
37 Room temperature
?. [Size selection on initial insertion library to generate ORF insertion library]
Purify the ligated DNA library.Use a single column and elute all DNA in .It is not necessary to quantify the DNA concentration.
[nuclease free water]
?. [Electroporation and library complexity quantification for ORF insertion library]
From this point onward, it is generally acceptable to move away from expensive commercial electrocompetent cells to in-house prepared electrocompetent cells.  The procedures are therefore nearly identical to the steps from   to   with minor changes. To make the protocol easier to follow, the steps are reproduced below with the changes incorporated.
?. [Electroporation and library complexity quantification for ORF insertion library]
The rest of the steps including cell scrapping and midiprep are the same.
?. [Substitution of transposon with split intein]
Take DNA from midipreped ORF insertion library, mix with the substitution insert in a 1:5 for library : insertion ratio and add to Golden Gate Assembly reaction.The substitution insert is provided in excess to increase the chance that all transposon within the library is replaced.Set up 6 *   in total.Enzymes used are ,  , andNote: the maximum amount of ORF insertion library DNA per 20 μL of Golden Gate reaction mix is 60 ng.  ABC1Number of reactions16210X T4 Ligase Buffer2123BbsI-HF164SapI165T4 DNA Ligase166ORF insertion library (452 ng/μL)0.1330.7987substitution insert (42 ng/μL)2.615.68water12.26773.602Aliquot the master mix into 2 * .Place them on thermocyclers and run the following program:  ABCD1StepTemperature (°C)Time21Digestion373 mins32Ligation164 mins4Repeat steps 1 - 2 for 25 cycles53Final Digestion3730 mins64Heat Inactivation6520 mins75Storage16infinite This protocol typically takes .
[of Golden Gate Assembly reaction]
ABC1Number of reactions16210X T4 Ligase Buffer2123BbsI-HF164SapI165T4 DNA Ligase166ORF insertion library (452 ng/μL)0.1330.7987substitution insert (42 ng/μL)2.615.68water12.26773.602
[ of reactions]
ABCD1StepTemperature (°C)Time21Digestion373 mins32Ligation164 mins4Repeat steps 1 - 2 for 25 cycles53Final Digestion3730 mins64Heat Inactivation6520 mins75Storage16infinite
?. [Preparation of DNA materials]
Prepare the linear substitution insert DNA
?. [Preparation of DNA materials]
Grow and harvest  for the plasmid carrying the substitution insert.Perform a midi-prep using the next day.
[bacterial culture]
?. [Preparation of DNA materials]
Digest from midiprep using and   ABC1NEB buffers and enzymes110210X CS Buffer2203DNA (819 ng/μL)1.2124BbsI-HF (20U/μL)1105SapI (10U/μL)1106ddH2O14.8148Note: DO NOT try to be thrift with SapI. It will result in incomplete digestion.Run reaction at  , for at least  , or  if time is permitted.
[substitution insert containing plasmid]
ABC1NEB buffers and enzymes110210X CS Buffer2203DNA (819 ng/μL)1.2124BbsI-HF (20U/μL)1105SapI (10U/μL)1106ddH2O14.8148
37 °C
?. [Electroporation and library complexity quantification for ORF insertion library]
Electroporate the ligated, ORF insertion DNA library into in-house electrocompetent cells.
?. [Electroporation and library complexity quantification for ORF insertion library]
Aliquot 2 *   into microcentrifuge tubes or 30 mL universal tubes / test tubes and warm up the medium in tubes at  for at least.
[SOC medium]
37 °C
?. [Electroporation and library complexity quantification for ORF insertion library]
Thaw and then mix   with the eluted DNA library in a pre-chilled microcentrifuge tube. Place the tube on ice for
[electrocompetent cells]
?. [Electroporation and library complexity quantification for ORF insertion library]
Aliquot    into a Ensure that the mixture is loaded in the gap. Place the cuvette on ice for
[DNA-competent cell mixture]
?. [Electroporation and library complexity quantification for ORF insertion library]
Take out the warmed up SOC medium.Wipe down any traces of water on the cuvettes. Carry out electroporation using Use protocol EC1. Between each cuvette, jot down the time constants for electroporation event. The value should be between 4 to 5.9 ms. Even if the value is out-of-range, keep going.Immediate after electroporation, add  into the electroporation cuvette. Gently suspend cells by mixing up and down for 1-2 times. Transfer all suspended cells in medium into the microcentrifuge or universal tube where the medium was warmed. For samples of the same library, pool the suspended cells together.It may be helpful to do this aseptically, but this is not strictly required. Speed is more important.
[SOC]
?. [Electroporation and library complexity quantification for ORF insertion library]
After finishing the electroporation steps for all samples, incubate the tubes at   for recovery.
37 33
?. [Electroporation and library complexity quantification for ORF insertion library]
Retrieve the recovered cells.For each library, there should be ~ 2 mL of recovered cells. Take out  and dilute it into  . Serially dilute it again 10-fold for 5 times. Label the tubes from A (lowest dilution) to E (highest dilution).Spread   onto 90 mm LB agar + KAN + TET plates labelled A to E. This is to quantify the "library complexity".Note that the antibiotics used change here to reflect to selection of different genetic components. TET is for selecting the protein expression vector, it may change if you use a different protein expression vector. KAN is for selecting the transposon.
[recovered cells]
[of 0.85% saline]
[diluted cells]
?. [Electroporation and library complexity quantification for ORF insertion library]
For the rest of the recovered cells, spread onto 2 * 145 mm LB agar + KAN + TET plates, each using ~ 1 mL of the cells. Allow the plates to dry before inverting them upside down.Incubate all plates overnight at
37 °C
?. [Electroporation and library complexity quantification for ORF insertion library]
Retrieve the plates and determine library complexity.For each library, tally the colony counts from plates A - E and create a table like below:If the library fold coverage is > 100. Proceed. Otherwise, discard the library.
ABCD1Plate# of coloniesdilution factorInitial number of transformants2ATMTC400-3B10840004320004C13400005200005D14000004000006E040000000Total number of colonies (aka library complexity) was 4.32 * 105.Size of trimmed CDS is 767 bp.Therefore, library fold coverage was 4.32 * 105/ ((767- 1) * 2) = 282 foldNOTE: when calculating library fold coverage for the ORF insertion library, the formula becomes:coverage = number of colonies / ((size of transposition window) * 2) the size of transposition = size of the trimmed CDS -1
?. [Substitution of transposon with split intein]
Pool all the Golden Gate Assembly reaction mixtures together and purify the final library.Use a single column and elute all DNA in .It is not necessary to quantify the DNA concentration.
[nuclease free water]
?. [Electroporation and library complexity quantification for the final library]
Repeat   to  .Note: if the target protein needs a reporter plasmid to manifest its function, then it is necessary to make bespoke electrocompetent cells that carry the reporter plasmid.
?. [Electroporation and library complexity quantification for the final library]
After the recovery step, there should be ~ 2 mL of recovered cells per library. Take and save as a glycerol stock.
[ recovered cells]
?. [Screening for AND / NAND gate behavior]
There is no fixed way to screen the final library. There could be different ways in which steps of enrichment or selection are conducted. Yet, in any case, the objective is to first isolate single colonies from the library that clearly show restoration of function when both the N- and C-lobes are expressed. The following steps provide a general description of what happens next.
?. [Electroporation and library complexity quantification for the final library]
Continue with   and   to estimate library complexity for the final library.
?. [Screening for AND / NAND gate behavior]
Once single colonies that show restoration of function are obtained, they are picked into 96-well plates with medium supplemented with appropriate antibiotics. Typically, > 500 colonies are picked.If your lab uses TipOne tips, the 3D printable Colony Picking Assistance might come in handy.
?. [Screening for AND / NAND gate behavior]
Grow the plates at  (overnight) to give a saturated culture.
37 33
?. [Screening for AND / NAND gate behavior]
Dilute   into 96-well plates with   supplemented with appropriate antibiotics and with 4 different combinations of inducers.Assuming the inducer  drives expression of the N-lobe and the inducer  (DAPG) drives expression of the C-lobe, the 4 combinations would bewater + DMF (solvent for DAPG)arabinose + DMFwater + DAPGarabinose + DAPGGrow inoculated plates at . The end time of this screening assay depends on the experiment context.
[overnight culture]
[medium]
37 33
?. [Screening for AND / NAND gate behavior]
Assay the plates and identify clones that restore the original protein function when both inducers are present.Cherry-pick and consolidate the individual wells into a new 96-well plate. Usually 93 - 195 colonies are good enough for subsequent steps.Using a robotic liquid handler like OT-2 is strongly recommended, otherwise you risk transferring the wrong wells because this task is prone to mental exhaustion and hence mistakes.
?. [Screening for AND / NAND gate behavior]
Grow the consolidated plate(s) at . This plate is now the stock. Duplicate this plate and make a 96-well  glycerol stock plate by adding  to . Freeze immediately in
37 33
-80 °C
[bacterial culture]
[50% glycerol]
[freezer]
?. [Screening for AND / NAND gate behavior]
Repeat   to   on the isolated clones for a rigorous experiment.Clones might behave different from the first time they are screened out. Keep only the clones that give consistent behavior.
?. [Whole-cell PCR and sequencing to identify split sites]
Once the clones are confirmed to show consistent AND or NAND behavior, proceed to whole-cell PCR.You only needs to amplify and sequence the region where the intein-extein CDS region join together. Both the N- or C- junction could be sequenced. In most cases the analysis is easier if the inteinC-exteinC junction is sequenced. This option is assumed for the steps below.
?. [Whole-cell PCR and sequencing to identify split sites]
Design and order primersThe forward primer should anneal ~ 50 - 100 bp before the 3' end of the inteinC CDS and the reverse primer should anneal  ~ 50 - 100 bp after the 3' end of the permitted transposition window.While designing primers, estimate the maximum size possible for the PCR product.
?. [Whole-cell PCR and sequencing to identify split sites]
Carry out whole-cell PCR using This is equivalent to colony PCR just that bacterial cells were obtained from liquid cultures instead of solid medium.Assuming you have 93 candidate clones to sequence, prepare 2 tubes of the master mixes below, each in a 1.5 mL microcentrifuge tube:  ABC1Number of reactions152210X ThermoPol Buffer2.513031mM dNTP52604FW primer0.5265RV primer0.5266Template bacteria0.5-7Taq Pol0.1256.58water15.875825.5Aliquot  into each well of a 96-well PCR plate, then add  to the  wells. There is no need to mix by pipetting up and down, which introduces bubbles. Seal the plate with an .Run the following protocol  ABCD1StepTemperature (°C)Time21Initial Denaturation952 mins32Denaturation9515 secs43Annealing5115 secs54Extension681 mins6Repeat steps 2 - 4 for 35 cycles75Final Extension685 mins86Storage12infinite
ABC1Number of reactions152210X ThermoPol Buffer2.513031mM dNTP52604FW primer0.5265RV primer0.5266Template bacteria0.5-7Taq Pol0.1256.58water15.875825.5
[PCR master mix]
[bacterial culture]
ABCD1StepTemperature (°C)Time21Initial Denaturation952 mins32Denaturation9515 secs43Annealing5115 secs54Extension681 mins6Repeat steps 2 - 4 for 35 cycles75Final Extension685 mins86Storage12infinite
?. [Whole-cell PCR and sequencing to identify split sites]
Resolve  on agarose gel to affirm PCR is successful.
[PCR product]
?. [Whole-cell PCR and sequencing to identify split sites]
Purify the PCR product.Elute DNA from each sample in .Dilute the eluted DNA to appropriate concentrations (10 ng/μL for Source BioSciences) and send to sequencing.Note: this step can be done manually but it is best to be facilitated using a robotic liquid handler.
[nuclease free water]
?. [Whole-cell PCR and sequencing to identify split sites]
Customize the Python scripts from our Github repository and analyze the sequencing data to identify split sites for each candidate clones. Merge split site information and protein activity data to produce an intein-bisection map.
?. [Preparation of DNA materials]
Purify the DNA.Load at most 2 gel slices (1 slice per lane on gel) per column.Elute DNA from each column using Pool eluted DNA together and quantify the concentration.
[nuclease free water]
?. [Preparation of DNA materials]
Resolved digested DNA on 1% agarose gel. Load   per lane.
[digested DNA]
Excise the band with size of 2591 bp, which corresponds to the substitution insert.