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Thaw BL21(DE3) Cells on ice
Pipette 50 μL of cells into transformation tube
Add 1 μL of protein encoded plasmids to transformation tube, flick 4-5 times
1. 170 ng/μl for R52-mCh-FL-BST
2. Unknown concentration for R52-mCh-H10-BST
Place mixture on ice for 30 mins.
1. Begin to warm up LB Broth
Heat shock at exactly 42°C for 10 secs.
1. Used water bath
2. Actual time: ~15 seconds
Place on ice for 5 mins.
Pipette 950 μL of room temperature LB Broth into mixture.
Incubate at 37°C with 250 rpm for 60 mins.
1. Warm selection plates to 37°C at the 30 min mark. (KAN resistance)
Mix cells thoroughly and spread 100 μL of the mixture onto a plate
1. One plate for either plasmid
2. Leftover transformation mixtures stored in 4°C fridge
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: Thaw BL21(DE3) Cells on ice 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 163 pg/μl for pUC19 Place mixture on ice for 30 mins. Begin to warm up LB Broth Heat shock at exactly 42°C for 45 secs. Used heat block instead of water bath Place on ice for 5 mins. Pipette 950 μL of room temperature LB Broth into mixture. Incubate at 37°C with 225 rpm for 60 mins. Warm selection plates to 37°C at the 30 min mark. (KAN resistance) 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 Incubate overnight at 37°C

expression

lysis and immobilization

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@@ Line 55: / Line 55: @@
 === [[Storage buffer]] ===
 === [[transformation]] ===
+=== Transformation of R5(2)-mCh-FL-BST and R5(2)-mCh-H10-BST ===
+==== protocol: ====
+#Thaw BL21(DE3) Cells on ice
+#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
+#Place mixture on ice for 30 mins.
+##Begin to warm up LB Broth
+#Heat shock at exactly 42°C for 10 secs.
+##Used water bath
+##Actual time: ~15 seconds
+#Place on ice for 5 mins.
+#Pipette 950 μL of room temperature LB Broth into mixture.
+#Incubate at 37°C with 250 rpm for 60 mins.
+##Warm selection plates to 37°C at the 30 min mark. (KAN resistance)
+#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
+#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:
+Thaw BL21(DE3) Cells on ice
+Pipette 50 μL of cells into transformation tube
+Add 1 μL of protein encoded plasmids to transformation tube, flick 4-5 times
+ng/μl for R52-mCh-FL-BST
+Unknown concentration for R52-mCh-H10-BST
+pg/μl for pUC19
+Place mixture on ice for 30 mins.
+Begin to warm up LB Broth
+Heat shock at exactly 42°C for 45 secs.
+Used heat block instead of water bath
+Place on ice for 5 mins.
+Pipette 950 μL of room temperature LB Broth into mixture.
+Incubate at 37°C with 225 rpm for 60 mins.
+Warm selection plates to 37°C at the 30 min mark. (KAN resistance)
+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
+Incubate overnight at 37°C
 === [[expression]] ===
 === [[lysis and immobilization]] ===

Revision as of 15:08, 4 September 2019

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Transformation of R5(2)-mCh-FL-BST and R5(2)-mCh-H10-BST

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