Contents 1 Introduction 2 Step1. Endonuclease digest PX330, pBx-093 2.1 Objective 2.2 Materials 2.3 Method 2.3.1 Endonuclease reaction system 2.3.2 Protocols 2.3.3 Supposed Results 2.4 Results 2.4.1 Laboratory note 3 Step2. Electrophoresis 3.1 Object 3.2 Materials 3.3 Method 3.3.1 Electrophoresis system 3.3.2 Protocols 3.3.3 Supposed Results 3.4 Results 3.4.1 Laboratory note 3.4.2 Laboratory result 4 Step3. Recovery of DNA from gell 4.1 Objective 4.2 Materials 4.3 Method 4.3.1 Protocols 4.4 Results 4.4.1 Laboratory note 4.4.2 Laboratory result 5 Step4. DNA Ligation 5.1 Objective 5.2 Materials 5.3 Method 5.3.1 Ligase reaction system 5.3.2 Protocols 5.3.3 Supposed Results 5.4 Results 5.4.1 Laboratory note 6 Step5. Transformation DH5α, Extract plasmid 6.1 Objective 6.2 Materials 6.3 Method 6.3.1 Protocols 6.3.2 Supposed Results 6.4 Results 6.4.1 Laboratory note 6.4.2 Laboratory result 6.4.3 Discussion 7 Step6. Transformation, endo free extract 7.1 Objective 7.2 Transformation 7.2.1 Materials 7.2.2 Method 7.2.2.1 Protocols 7.2.3 Result 7.2.3.1 Laboratory note 7.2.3.2 Laboratory result 7.3 Endo-free extract 7.3.1 Materials 7.3.2 Method 7.3.2.1 Procedure 7.4 Results 7.4.1 Laboratory note 8 Step7. Verification of plasmid 8.1 Objective 8.2 Materials 8.3 Protocol 8.4 Supposed Results 8.5 Results 8.5.1 Laboratory note

PB5-TetOn-Cas9-PB3 plasmid construction

2014/7/27 Construct a plamid to stably transfect HeLa cells!

Introduction

This is the notebook about constructing one of the most important plasmids, which is PB5-Puro-TetOn-Cas9-PB3(or Cas9+Tet). Cas9+Tet will be statbly tranfected into HeLa cells and express Cas9 protein induced by [http://en.wikipedia.org/wiki/Doxycycline Doxycycline](It is in the tetracycline antibiotic class).

Figure 1. Schematic of the plasmid. PB5-PB3 is the transponase traget sequence. TetON 3G is a protein induce pTRE-3G expression when bind with doxycycline. 

Step1. Endonuclease digest PX330, pBx-093

Objective

• Digest the plasmids for cloning new plasmid

Materials

• Plasmid: px330, pBx-093

• Endonuclease: AgeI, NotI, AflIII (NEB)

• Endonuclease Buffer: NEBuffer 2.1

• Some EP tube

• Water bathe

Method

Endonuclease reaction system

• AgeI + NotI + AflIII digest px330

• AgeI + NotI digest pBx-093

• Time: 5hours
• Temperature: 37°C
• Heat inactivation: 70°C 20min

Protocols

Procedure: Digestion

1. Prepare all the materials, placing enzyme in ice, two EP tube marking A and B
2. Pipet every component in the list and follow the oreder
   • In A tube
     1. 9μl dd₂O
     2. 2μl NEBuffer 2.1
     3. 6μl Plasmid px330
     4. 1μl AgeI
     5. 1μl NotI
     6. 1μl AflIII
   • In B tube
     1. 11μl dd₂O
     2. 2μl NEBuffer 2.1
     3. 5μl plasmid pBx-093
     4. 1μl AgeI
     5. 1μl NotI
3. Incubate in 37°C for 5 hours
4. After 5 hours 70°C water bath for 20min to inactivition

Supposed Results

• For pBx-093
  1. PB5-Puro-TetOn-PB3 vector: 7708bp
  2. Fragment: 2313bp

• For px330
  1. Cas9: 4523bp
  2. Fragment * 2: 1244bp + 2739bp

Results

Laboratory note

[2014 Aug 5th 15:20pm]: Started adding all compotent according to Protocols

[2014 Aug 5th 15:30pm]: Started incubation in 37°C

[2014 Aug 5th 20:30pm]: 70°C water bathed EP tubes for 20min

[2014 Aug 5th 20:50pm]: Placed the EP tubes in ice wait for electrophoresis

Step2. Electrophoresis

Object

Seperate DNA fragment by its atom mass, so we can purify and isolate the vector and insert DNA which we need.

Materials

• DNA loading buffer

• Marker dl5000(Takara)

• Marker dl15000(Takara)

• Gel green DNA dye

• TAE buffer:

• Argarose 1%

• TAE buffer

Method

Electrophoresis system

voltage: 100V time: 40min

Protocols

Procedure of electrophoresis

1. Argarose gel making
   1. take 0.5g argarose
   2. dissovle in argarose in 50ml TEA buffer
   3. heating to dissolve for 1.5min
   4. add GelGreen*(Tiangen) 1μl
   5. pour the melting gel in mold
   6. cool down for 20min for solidification
2. Load DNA Sample and marker on the gel
3. Electrophorese at 100Volt for 45minutes

Supposed Results

Due to the complete digestion, DNA sample will be seperated in to few strips on the electrophoresis image representing different DNA size.

• pBx-093

Two strips at 7708bp(we need) and 2312bp

• px330

Three strips at 4523bp(we need), 2734bp, 1244bp

Results

Laboratory note

[2014 Aug 5th 20:35pm] During enzyme inactivation, we made the ararose gel and poured into mold.

[2014 Aug 5th 20:55pm] Put gel into electrophoresis chamber drown with TEA buffer, set up all parameter of electrophoresis power.

[2014 Aug 5th 20:57pm] Loaded sample in the order of following table

[2014 Aug 5th 21:10pm] Started electrophoresis

[2014 Aug 5th 21:55pm] Took photo of gel.

Laboratory result

Figure 2. The image of electrophoresis of digestion product of px330 and pBx-083 with NotI, AgeI, AflIII. The first three columes are px330 react with AgeI and NOtI, The other columes are pBx-093 digest with AgeI and NotI and AflIII(additional cut for fragment seperation). Both the first row are strips we need.

The image of electophoresis shows that all strips are beautifully bright and clear and straight, moreover, on the exact position relative to marker. Which means that the endonuclease digestion and electrophoresis are successful.

The first two steps are perfect for next step.

Step3. Recovery of DNA from gell

Objective

After electrophoresis, cut the correct strip, recover and purify DNA from the strip to get the exact DNA fragement we need.

Materials

• DNA Argarose recovery kit(TIANgel Midi Purification kit)

• EP tubes

• Dry heater

• Gel strip

Method

Protocols

Procedure:

1. Cut the strip form argarose gel
2. Recover DNA, with recovery kit. Follow the kit protocol.

Results

Laboratory note

[2014 Aug 5th 22:00pm] Started DNA recovery.

[2014 Aug 6th 1:00pm] Finish DNA recovery and measure concentration of DNA solution

Laboratory result

• Vector PB5-Puro-Tet-PB3 : 9.3ng/μl
• Inster Cas9 protein gent: 5.7ng/μl

The concentration of DNA is good for next step.

Step4. DNA Ligation

Objective

• Recombinant DNA from the recovered DNA fragment, to construct new plasmid

Materials

• Insert DNA: Cas9 4523bp 5.7ng/μl
• Vector DNA: PB-Tet 7708bp 9.3ng/μl
• T4 ligase and buffer (NEB)
• EP tubes
• EP tube heater

Method

Using the T4 ligase to ligand the two DNA fragement to construct the new plasmid

Ligase reaction system

From the protocol of T4 ligase we the mole ratio of Vetor to Insert is 1:5. So after integral the data of the numbers base pair of two DNA and concentration, we get that the volume ratio of vector to insert is 1 : 5.3
To get the reliable data and test if vector and ligand its self, we set a negertive control group 3. We also set two parallel control group 1 and 2 The ligation reaction system

Reaction Condition

37°C

Total volume

20μl

Time

20min

Heat inactivation: 65°C 10min

Protocols

Procedure: Ligation

1. Prepare 3 EP tubes, marking 1,2,3.
2. Add PB-TetOn DNA 2.7μl to all tubes.
3. Add Cas9 DNA 14.3μl to tube1 and tube2.
4. Add 14.3μl ddH₂O to tubes 3.
5. Add 2μl buffer to all tubes.
6. Add 1μl T4 DNA Ligase to all tubes
7. Place all tubes in to incubator at 37°C for 20min
8. After 20min, water bath all tubes at 65°C for 10min
9. Store all tubes at -20°C

Supposed Results

• In group 1 and 2, there should be circular and correct plasmid
• For negative control goupe all DNA should be linear

Results

Laboratory note

[2014 Aug 6th 10:00am] Start prepare all materials and add all reactent. [2014 Aug 6th 10:12am] Place the system in incubator at 37°C [2014 Aug 6th 10:32am] After 20min incubation, inactivation place all tubes on ep tube heater at 65°C for 10min. [2014 Aug 6th 10:42am] Store the ligation product at -20°C waiting for next step.

Step5. Transformation DH5α, Extract plasmid

Objective

Materials

• Ligation production 3 groupes

• Ice box

• EP tubes

• Competent cell: DH5α

• LB agar plate with Amp. antibiotics

• 42°C waterbath

• LB media

• Plasmid mini extract kit(TianGen)

• Pipette tips

Method

Transform the Ligation production into competent cell. All the linear DNA will not have any impact to cell and curcular plasmid may transform into competent cells. Those cell with plasmid can express the resistence to Amp. because the vector PB5-TetON-PB3 has a resistence gene of Amp.
After bacteria on plate grows into a colony, we pick up the colony inoculate into LB and incubation over night to amplificate the plasmid.

Protocols

Procedure: Transformation

1. Thaw competent cells on ice and then pre-chill by placing the tubes on ice.
2. Pipet 50μl of competent cells into 3 EP tubes.
3. Pipet 1μl of ligation product DNA into each tube. Flick the tube gently to mix.
4. Incubate on ice for 30 minutes.
5. Heat-shock the cells, 42°C waterbath, 1 minute.
6. Immediately transfer the tubes back to ice, and incubate on ice for 2 minutes.
7. Add 300ul of LB media per tube, 37°C, 45 min at 180rpm.
8. Prepare 4 argar plates marking 4 different groups (One without plasmid)
9. Pipet 20ul from each tube, spread the mixture evenly across the plate.
10. We also did a control . Except for not being transfected by plasmid, other Protocolss were all the same with those experimental groups.
11. Incubate these agar plates at 37°C overnight.

Procedure: Plasmid Extract

1. Pick monoclones with tweezers and a sterile white pipette tip.
2. Put the tip into 3ml LB in test tube.
3. Incubate test tube at 37°C over night
4. Extract plasmid following the protocol of plasmid extract kit
5. Concentration test with nanodrop

Supposed Results

• Clones on plates of Group1 and Group2.
• No clones on Group3 and blank.
• Can extract correct plasmid from monoclones.

Results

Laboratory note

[2014 Aug 6th 11:00am] Start transformation

[2014 Aug 6th 11:37am] Start ice icubation and have launch

[2014 Aug 6th 12:11am] Add LB, place tubes in 37°C

[2014 Aug 6th 13:00pm] Spread the mixture on plate with antibiotic Amp.

[2014 Aug 6th 13:14pm] Place all plate in 37°C .

After 21hours [2014 Aug 7th 10:00am] Picks 6 colnies and shake in 3ml LB at 37°C.

After 12hours [2014 Aug 7th 22:00pm] Plasmid extract

Laboratory result

Group 1: 36 colonies Group 2: 90 colonies Group 3: 18 colonies Blank  : 0 colonies

Figure 3. Picture of group 1, with 36 clones.

Figure 4. Picture of group 2, with 90 clones.

Figure 5. The strange result of negative control, supposed to be no colonied. But the plate has 18 colonies.

Discussion

• There are 18 colones on the negative control plate. But all the DNA ligation ractant are recover from gel. So the fake positive result probably comes from the complemental last two nucleus tide of sticky end of AgeI and NotI.
  
• To avoid picking the fake positive plasmid we choose 6 monoclones 2 form group 1 and 4 frome group 2. Then it is almost impossible to fail to extract the correct plasmid.

Step6. Transformation, endo free extract

Objective

Transformation

Materials

• Plasmid number:1.2

• Ice box

• EP tubes

• Competent cell: DH5α

• LB agar plate with Amp. antibiotics

• 42°C waterbath

• LB media

Method

Protocols

Procedure

1. Thaw competent cells on ice and then pre-chill by placing the tubes on ice.
2. Pipet 50μl of competent cells into 1 EP tube.
3. Pipet 1μl of plasmid into tube. Flick the tube gently to mix.
4. Incubate on ice for 30 minutes.
5. Heat-shock the cells, 42°C waterbath, 1 minute.
6. Immediately transfer the tubes back to ice, and incubate on ice for 2 minutes.
7. Add 300ul of LB media into tube, 37°C, 45 min at 180rpm.
8. Prepare 1 argar plate marking "Cas9+TetOn"
9. Pipet 20ul from tube, spread the mixture evenly across the plate.
10. Incubate these agar plates at 37°C overnight.

Result

Laboratory note

[2014 Aug 7th 9:00am] Start transformation, thaw competent cell

[2014 Aug 7th 9:07am] Added plasmid, start ice icubation

[2014 Aug 7th 9:38am] Heat shock, ice stimulate, add LB

[2014 Aug 7th 9:41pm] Place the tube with cell in 37°C for 45min

[2014 Aug 7th 10:30pm] Spread the mixture on plate with antibiotic Amp.

[2014 Aug 7th 10:34pm] Place all plate in 37°C.

Laboratory result

After 12hours incubation in 37°C plate is full of colonies, plasmid is partially successful and transformation may have use plasmid with too high concentration.

Endo-free extract

The plasmid has a resistance of ampicillin, so only those have been transformed with a plsmid can grow. After bacteria on plate grows into a colony, we pick up the colony inoculate into LB and incubation over night.

Materials

• Plasmid midi endofree extract kit(Tiangen)
• 30ml LB broth
• Pipette tips

Method

We pick up the clone and inoculate in 30ml LB broth to amplificate plasmid in order to get high concentration plasmid solution.

Procedure

1. Pick monoclones with tweezers and a sterile white pipette tip.
2. Put the tip into 30ml LB in test tube.
3. Incubate test tube at 37°C over night
4. Extract plasmid following the protocol of endo-free plasmid extract kit
5. Concentration test with nanodrop

Results

Laboratory note

[2014 Aug 8th 10:30pm] Pickup clones

[2014 Aug 8th 10:30pm] Inoculate clones into 30ml LB.

[2014 Aug 9th 10:30am] Endo-free extract plasmid

Step7. Verification of plasmid

After extracting plasmid we have to verify the plasmid. We have to know wehther the plasmid is the same as we what or we have some truble.

Objective

• Digest the plasmid and electrophorese for verification

Materials

• Endonuclease: PstI(NEB), EcoRI(NEB), EcoRV(NEB), ApaI(NEB)
• Buffer: NEBuffer 3.1, CutSmart(NEB)
• Plasmid: Endofree Cas9+Tet
• ddH₂O

Protocol

Incubation condition: 25°C 20min

Heat inactivation: 65°C 20min

Incubation condition::37°C 60min

Heat inactivation: 65°C 20min

37°C 20min

Heat inactivation: 80°C 20min

After electrophoresing the product of digestion

Supposed Results

Results

Laboratory note

Figure 6. All six plasmid has digest. The strips of 1422 1351 1212 are the characteristic strips. Digestion can generate such strips only if Cas9 is inserted into TetON vector.

The image of electrophoresis of PstI digestion of first amplicated. Six plasmids of different six monoclone are form two test group insert with Cas9 proterin into TetOn vector. The image shows that the strip of electrophoresis is kind of dim, but straight and clear, which is at the position it suppose to be relative to marker.

Some strips went out the gel, so we can not see those from the image. The cause of out range may be high voltage and too long running time. So we low down the voltage and cut the running time in the following futhur verification.

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Endo-free plasmid verification

Figure 7. Endo free extracted plasmid have endonuclease digest, and the product is eletrophoresed. Colume 1 stands for restriction digestion of ApaI. Colume 2 stands for EcoRI and EcoRV. Colume 3 stands for PstI digestion result.

Figure 8. Reverification of nuclease digestion. A is product of restriction digest with ApaI, E is EcorI+EcorV, P is PstI.