Transformation of R5(2)-mCh-FL-BST and

Taken from the lab notebook of Dushanth Seevaratnam

protocol:

1. Thaw BL21(DE3) Cells on ice
2. Pipette 50 μL of cells into transformation tube
3. Add 1 μL of protein encoded plasmids to transformation tube, flick 4-5 times
   • 170 ng/μl for R52-mCh-FL-BST
   • Unknown concentration for R52-mCh-H10-BST
4. Place mixture on ice for 30 mins.
   • Begin to warm up LB Broth
5. Heat shock at exactly 42°C for 10 secs.
   • Used water bath
   • Actual time: ~15 seconds
6. Place on ice for 5 mins.
7. Pipette 950 μL of room temperature LB Broth into mixture.
8. Incubate at 37°C with 250 rpm for 60 mins.
   • Warm selection plates to 37°C at the 30 min mark. (KAN resistance)
9. Mix cells thoroughly and spread 100 μL of the mixture onto a plate
   • One plate for either plasmid
   • Leftover transformation mixtures stored in 4°C fridge
10. Incubate overnight at 37°C

Results: The selection plates showed no colonies.

Troubleshoot: Increase heat shock time from 10 seconds to 45 seconds Additional transformation with pUC19 Plate the transformed cells with non-selection Agar plates along side selection plates Use heat block instead of water bath

Second Attempt - Protocol:

1. Thaw BL21(DE3) Cells on ice
2. Pipette 50 μL of cells into transformation tube
   • Add 1 μL of protein encoded plasmids to transformation tube, flick 4-5 times
   • 170 ng/μl for R52-mCh-FL-BST
   • Unknown concentration for R52-mCh-H10-BST
3. 163 pg/μl for pUC19
4. Place mixture on ice for 30 mins.
   • Begin to warm up LB Broth
5. Heat shock at exactly 42°C for 45 secs.
   • Used heat block instead of water bath
6. Place on ice for 5 mins.
7. Pipette 950 μL of room temperature LB Broth into mixture.
8. Incubate at 37°C with 225 rpm for 60 mins.
   • Warm selection plates to 37°C at the 30 min mark. (KAN resistance)
9. Mix cells thoroughly and spread 100 μL of the mixture onto a plate
   • One Agar plate w/ KAN for BST plasmids
   • One Agar plate w/ AMP for pUC19
   • One Agar plate w/o KAN for all 3 plasmids
   • Leftover transformation mixtures stored in 4°C fridge
10. Incubate overnight at 37°C

Results: The selection plates showed no colonies. Both KAN (BST) and the single AMP (pUC19) plate had no signs of cell growth. On the other hand, all the non-selection plates had plenty of cell growth.

Possible reasons: BL21 (DE3) cells have lost their competency The lack of SOC media is causing the cells to lose any plasmids taken in before full recovering from the heat shock

Troubleshoot: Replace BL21 (DE3) cells with BL21 (DE3)pLysS from another lab. Follow the competence and transformation protocol used by the other lab Prepare SOC media and use it after heat shock instead of LB Broth

Chemicals for BL21(DE3) pLysS transfromation protocol

1. 1 M MgCl2
   • Weigh 1.9042g of MgCl2 anhydrous and mix with 20 ml sterile Milli-Q water.
   • Sterilize by passing through 0.22 μm filter
2. 1 M CaCl2
   • Weigh 2.2196g of CaCl2 anhydrous and mix with 20 ml sterile Milli-Q water.
   • Sterilize by passing through 0.22 μm filter

Prepare MgCl2 + CaCl2 mixture for transformation

Chemicals	Volume
1 M MgCL2 (filter sterilized)	4 ml
1 M CaCl2 (filter sterilized)	1 ml
Sterile Milli-Q water	45 ml

Prepare BL21 (DE3) pLysS competent cells

1. Incubate at 37°C, 200 rpm for 2 to 3 hours.
   • Actual duration: 2 hours 30 minutes
2. Transfer culture into four 2 ml tubes (2 ml each tube) on ice.
3. Spin down cells at 6,400 rpm, 4°C for 10 minutes.
4. Decant supernatant. Add in 2 ml of MgCl2 + CaCl2 mixture and resuspend gently.
5. Place on ice for 30 minutes.
6. Spin down cells at 6,400 rpm, 4°C for 10 minutes.
7. Decant supernatant and add 92 μl of 0.1 M CaCl2 and 4 μl of DMSO.
8. Place on ice for 15 minutes.
9. Add in 4 μl of DMSO.

A#liquot into Eppendorf tubes at 50 μl each tube and store in -80°C.

DNA transformation into BL21 (DE3) pLysS competent cells

1. Thaw BL21(DE3) pLysS cells on ice
2. Add 1 μL of protein encoded plasmids to transformation tube, flick 4-5 times
   • 170 ng/μl for R52-mCh-FL-BST
   • Unknown concentration for R52-mCh-H10-BST
   • 163 pg/μl concentration for pUC19
3. Place mixture on ice for 30 mins.
   • Begin to warm up SOC medium
4. Heat shock at exactly 42°C (water bath) for 40 secs.
5. Place on ice for 2 mins.
6. Pipette 250 μL of room temperature SOC medium into mixture.
7. Incubate at 37°C with 225 rpm for 90 mins.
   • Warm selection plates to 37°C at the 60 min mark. (2 KAN resistance, 1 AMP resistance)
8. Mix cells thoroughly and spread 150 μL of the mixture onto a plate
   • One Agar plate w/ KAN for BST plasmids
   • One Agar plate w/ AMP for pUC19
   • Leftover transformation mixtures stored in 4°C fridge
9. Incubate overnight at 37°C

Results The selection plates showed:

• 50-100 colonies for R52-mCh-H10-BST
• >300 colonies for R52-mCh-FL-BST