Team:ITB Indonesia/Notebook/WetLab

June 2013

Saturday, 1 June 2013
Making CCMB 80 buffer and CaCl2 + Gliserin 15% buffer.

Sunday, 2 June 2013
Making competent cell with iGEM standard method (CCMB 80 Buffer) and with CaCl2 + Gliserin 15% buffer.

Tuesday, 4 June 2013
Transform the plasmid to DH5α competent cell. We compared number of colony from CaCl2 method and  from iGEM standard method. For each method, we spread 20 µl and 200 µl culture.

Wednesday, 5 June 2013
Count the transform colony. Wehave found that the number of colony from 20 µl with CCMB 80 Buffer gave  a better result than 200 µl with CaCl2 + Gliserin 15% buffer. So, we are confident with iGEM standard method.

Thursday, 6 June 2013
We try varying the time and repetition of heat shock during transformation. We use single heat shock and double heat shock, and 60 sec and 90 sec for each method.

Friday, 7 June 2013
Count the transformation colony. Wehave found that the number of colony from single heat shock for 60 sec giving the best result than other. So, we applied this method for our transformation.

Saturday, 8 June 2013
Repeating transformation like a day before

Monday, 10 June 2013

• Transform xxx using competent cell and agar plate made of SOB, heat shock 60 seconds 42⁰C, duplo.

Tuesday, 11 june 2013

• Making SOC medium
• Transforming BBa_J04450 (pSB1C3) from transformation eficiency kit (concentration 5pg/ µl) with 1M IPTG induction : 0 µl, 2 µl, 4 µl
• Making 3ml DH5α  broth culture from gliserol stock
• Counting the transformacy efficiency from the day before. From first plate resulting 300 colony, and 258 colony from the second
• Discussing 3A assembly system and white screening selection
• Making LB agar stock

Thursday, 13 June 2013

• Making gliserol stock of BBa_J000450. The colonies grew
• Making glycerol stock of BBa_E0430. The colonies grew well.

Friday, June 14th 2013

• Transforming BBa_J22106. The colonies grew poorly
• Transforming BBa_J06702. The colonies grew well.
• Transforming BBa_I13507. The colonies grew well.
• Transforming BBa_I765001. The colonies grew poorly.
• Transforming BBa_E0840. The colonies grew well.

Friday, June 14th 2013

• Preparing solutions for plasmid isolation by alkali lysis method
• Making glycerol stock of 4B2 and 20K3
• Making liquid culture of 9N5, 16B3, 23C3

Monday, June 17th, 2013

• BBa_K592009
• BBa_E1010
• BBa_K592011
• BBa_K592010

Tuesday, June 18th, 2013

• Making liquid culture of 19E1 and 12N3
• Transforming BBa_B0015
• Transforming BBa_B1006
• Making 5X ligation buffer

Wednesday, June 19th, 2013

• Making stock culture of 19E1 and 12N3
• Plasmid isolation from 19E1 and 12N3 stock culture
• Making 1M Tris solution, pH 7.5
• Making 0.5M EDTA slution, pH 8.0

Thursday, June 20th, 2013

• Making stock culture of 4F3 and 6D3
• Plasmid isolation from 4F3 and 6D3 stock culture
• Making and sterilizing TE Buffer

Friday, June 21st, 2013

• Transforming BBa_K873002
• Trannsforming BBa_B0034

Saturday, June 22nd, 2013

• Making glycerol stock of 3H1
• Plasmid isolation from 3H1 stock culture
• Discussing the planned system

Sunday, 23rd, 2013

• Transforming BBa_J23119

Monday, June 24th, 2013

• Confirming results from plasmid isolation by gel electrophoresis

Tuesday, June 25th, 2013

• Plasmid isolation from 18D3 and 18A5 stock culture

Thursday, June 27th, 2013

• Plasmid isolation from 20K3, 16B3, 23C3, 21B5, 9N5, 19E1, 12N3, 4F3, 6D3, and 3HI stock culture
• Confirming results from plasmid isolation in June 25 and 27th by gel electrophoresis

Sunday, June 30th, 2013

• Inoculating (parts?) in 25mL of LB broth + 25 uL of antibiotic

July 2013

Monday, July 1st, 2013

• Transforming RBS 1 (strong) B0030. The colonies grew well.
• Gel electrophoresis of 16B3, 23C3, 21B5, 9N5
• Inoculating 4F3, 6D3, 3H1, 18D3, and 18A5 in 25mL of LB broth + 25 uL of antibiotic

Tuesday, July 2nd, 2013

• Plasmid isolation from 4F3, 6D3, 3H1, 18D3, and 18A5 stock culture using alkali-lysis buffer solution
• Making liquid culture from colonies in agar plate (?)
• Preparing solutons for restriction digest (1X NE Buffer 1, 1X NE Buffer 2, 1X NE Buffer 3, 1X NE Buffer 4)
• Confirming results from plasmid isolation by gel electrophoresis
• Restriction digest of 9N5 (Part A, SOS Promoter) and 23C3 (Part B, GFP Generator) and assembly using 3A method

Wedenesday, July 3rd, 2013

• Making NaCl 5M stock solution
• Gel purification of 4F3 and 3H1
• Restriction digest of 16B3 using E+P
• Confirming digestion results by gel electrophoresis. The resulting band was so thin.
• Restriction digest of 16B3 using E+X and 21B5 using E+S

Monday, July 8th 2013

• Restriction digest  of 4B2 using E+P, 23C3 using X+P, 21B5 using E+S, 9N5 using E+S.
• Assembling the digestion product using 3A method

Thursday, July 11th, 2013

• Plasmid isolation of pSBIK3
• Confirming isolation results by gel electrophoresis
• Restriction digest of plasmid backbone pSBIK3
• Ligation of SOS Promoter + GFP + pSBIK3
• Transforming ligation result into 50 uL of competent cells
• Confirming result from plasmid isolation (1G5, 2H2, 5A5, 6B2) by gel electrophoresis. No band appeared.

Saturday, July 13rd, 2013

• Inoculating pUV + GFP to LB broth
• Making stock culture of pSOS + GFP. Centrifugation result showed that the pSOS + GFP failed to insert into the backbone, hence the transformation, inoculation, and isolation steps was repeated.
• Ligation of pUV + GFP + pSBKI3 in 4oC overnight

Sunday, July 14th, 2013

• Centrifugation result showed that the pUV + GFP sucessfully inserted into the backbone
• Plasmid isolation of 2.1, 2.2, 2.3, and 2.4, stored in -20oC
• Confirming result from plasmid isolation by gel electrophoresis
• Amplified the result by PCR

Monday, July 15th, 2013

• Restriction digest of plasmid 2.3 using EcoRI and Pst I
• Confirming result from restriction digest by gel electrophoresis. The result showed that the digestion was failed.
• Plasmid isolation from pSOS + GFP + pSBIK3
• Preparing 200 mL of LB agar

Tuesday, July 16th, 2013

• Scaling up the pSBKI3 stock culture
• Confirming result from plasmid isolation of pSOSGK1, pSOSGK2, pSOSGK3, pSOSGK4, pSOSGK5 by gel electrophoresis.

Wednesday, July 17th, 2013

• Repeating the restriction digest using E+P
• Plasmid isolation from scale up culture of pSBKI3, pSBIA3

Thursday, July 18th, 2013

• Preparing 5 mL of liquid broth containing E. coli  BL21 (DE3)
• Plasmid isolation of 2.2 from stock culture
• Confirming result from plasmid isolation of 2.2 and restriction digest for pUV, 23C3, 9N5, 6B2 by gel electrophoresis. The result was failed.

Friday, July 19th, 2013

• Preparing 50 mL of liquid brith containing E. coli BL21 (DE3)
• Resuspending the culture in 70% ethanol
• Optimizing condition for restriction digest (4oC for 14-16 h vs 37oC for 4 h) using GFP, X, and P

Saturday, July 20th, 2013

• Ending the overight incubation
• PCR of 2.1, 2.3, and 2.4 (results from plasmid isoaltion of pUV+GFP) using VF2 and V2 primers.
• Gel electrophoresis of 2.1, 2.2, and 2.4. The result was failed possibly due to high annealing temperature (64oC).
• Repeating PCR of 2.1, 2.3, and 2.4 using lower temperatures (60oC) followed by gel electrophoresis. The result was successful.
• PCR of pSOSGK1, pSOSGK 2, pSOSGK3, pSOSGK4, pSOSGK5 followed by electrophoresis. The result was successful

Monday, July 22nd, 2013

• Restriction digest of 23C3, 2.1, 2.3, and 2.4 using X+P and buffer H
• Scaling up of 23C3 from glycerol stock
• Gel electrophoresis of 23C3, 2.1, 2.3, and 2.4
• Scaling up of pUV + GFP + pSBKI3 from glycerol stock

Tuesday, July 23rd, 2013

• Plasmid isolation of 23C3
• Scaling up of pUV + GFP
• Restriction digest of pSOS + GK1 using X+P
• Gel electrophoresis of pSOSGK4 uncut, 23C3, and scaled-up pUV + GFP

Thursday, July 25th, 2013

• Restriction digest of monomeric RFP (mRFP) BBa_E1010
• Ligation of pSOS + GFP + pSBIK3
• Transforming restriction and ligation results
• PCR of pSOSGK1, pSOSGK 2, pSOSGK3, pSOSGK4, pSOSGK5 followed by gel electrophoresis. The result was successful

Friday, July 26th, 2013

• Double restriction digest of pSOSGK1, pSOSGK 2, pSOSGK3, pSOSGK4, pSOSGK5, and pSBIK3 using X and then using P
• Making liquid culture of pSOS + GFP + pSBKI3, incubated for 18 h at 37oC.
• Gel electrophoresis of pSOSGK1, pSOSGK 2, pSOSGK3, pSOSGK4, pSOSGK5. The result was successful

Saturday, July 27th, 2013

• Gel electrophoresis of digestion results using X+P
• Gel electrophoresis of isolated pSOS + GFP

Sunday, July 28th, 2013

• Project presentation to Mrs. Betty

Monday, July 29th, 2013

• Repeating the restriction digest of pSOSGK1, pSOSGK 2, pSOSGK3, pSOSGK4, pSOSGK5, and pSBIK3
• Restriction digest of pSBIC3 plasmid backbone, incubated for 4 h at 37oC
• Ligation of pSBIC3 + pUV + GFP and pSBIC3 + pSOSGK5
• Repeating the gel electrophoresis for pSOSGK1, pSOSGK 2, pSOSGK3, pSOSGK4, pSOSGK5, and pSBIK3, including digestion result for pSBIC3
• Transforming the ligation results. The colonies didn’t grow.

Tuesday, July 30th, 2013

• Repeating the restriction digest of pSBIC3 from 12N3
• Restriction digest of pSBIC3 from BBa_J04450
• PCR of pSOS + GFP + pSBIK3 followed by electrophoresis
• Inoculating BBa_J04450 from glycerol stock to 25 mL of liquid broth
• Discussing about the next step of making dried E. coli cell
• Making 250 mL of LB broth

Wednesday, July 31st, 2013

• Measuring growth rate and plotting growth curve for dried E. coli cell

Thursday, August 1st, 2013

• Preparing solutions for plasmid isolation
• Making 250 mL of LB agar and 250 mL of LB broth
• Inoculating colonies of pSOS + GFP + pSBIK3
• Preparation for DNA sequencing

Friday, August 2nd, 2013

• Sending DNA sequence (2.4, pSOGK1, pSOGK5) to Macrogen
• Observing results from inoculation
• Preparing 5 mL of LB broth

Saturday, August 3rd, 2013

• Plasmid isolation until resuspension