Team:BostonU/Notebook

• We performed PCR to amplify pMJSAF using the following MoClo forward and reverse primers.
• DVL0_AF with DVL0_BR (MF1)
• DVL0_BF with DVL0_CR (MF2)
• DVL0_CF with DVL0_DR (SJ1)
• DVL0_DF with DVL0_ER (SJ2)

• We performed Gel Electrophoresis to confirm that pMJSAF was amplified in PCR.

• The gel ran for too long thus the bands ran off of the gel. which can be seen towards the bottom of the picture. We expect for the band to be around 500 bp

• We reperformed the PCR amplification of pMJSAF with the same pairs of primers:

• DVL0_AF with DVL0_BR (MF3)
• DVL0_BF with DVL0_CR (MF4)
• DVL0_CF with DVL0_DR (SJ3)
• DVL0_DF with DVL0_ER (SJ4)

• We performed Gel Electrophoresis on this set of DNA.
• gel 1 pic The amplification of pMJSAF displayed strong bands at 500 bp for the primers : DVL0_BF with DVL0_CR and DVL0_DF with DVL0_ER. However for the   DVL0_AF with DVL0_BR showed a much weaker band, and the DVL0_CF with DVL0_DR did not show at all.

• We gel extracted MF3,4 and SJ4. Using the gel extraction protocol.
• We then quantified the amount of DNA we had using NanoDrop DNA quantification protocol
• nanodrop 1

• We set up a restriction digest (SpeI, BSA, Buffer 4)for MF3,4 and SJ4. following the Restriction digest protocol
• We transformed iGEM plasmids with competent bacteria from stock, and plated them.  We grew them overnight in 37C incubator.
• pSB1A3 plate 1 well 1G  (1A3)
• pSB1AT3 plate 1 well 13A (1AT3)
• pSB1C3 plate 1 well 3A (1C3)
• pSB1K3 plate 1 well 5A (1K3)

• We thus performed gradient PCR  of SJ3 and MF3 in order to set the best parameters for the amplification (62-69 with interval of 1 degree C)
• gel2 pic
• We later found out, that our step 1 was set at 62C instead of 95C, which could have affected denaturation of the DNA template; although the SJ5-12 was amplified while the MF5-12 did not. We are unsure to the cause. The results were inconclusive compared to the Gel made previously.We ran another PCR to verify results.
• We did PCR clean up of MF3,4 and SJ4  
• We quantified the product of MF3,4 and SJ4 PCR clean up using NanoDrop
• nanodrop 2

• We were given new primers to make stock and working solutions from. In the process we realized that we used DVL1_ER instead of DVL0_ER in SJ4, thus all SJ4 products were discarded and data obsolete. We remade the DVL1_ER stock solution by adding additional EB buffer till the right concentration has been reached. We then proceeded to make stock and working solutions for:
• DVL0_ER,
• DVL1_AF
• DVL1_ER
• DVL1_FF
• DVL1_FR
• DVL1_GF
• DVL1_GR
• DVL1_HR

• Then we paired AF-ER, FF-GR, EF-FR, GF-HR to amplify the template DNA using PCR
• We picked colonies from the plates that were stored in the incubator and allowed the picked colonies to be incubated in a 37C shaker
• We made the gel from the previously mentioned PCR of AF-ER (MS4), FF-GR (MS5), EF-FR(MS6), GF-HR (MS7) and also 3 L0 (MF3=MS1, SJ3=MS2,  SJ4=MS3)
• gel3 pic
• ALl the L0 failed and the L1 showed bands
• We performed gel extraction on the 4 L1 primer based amplification
• We performed nanodrop quantification of the products of the gel extraction
• nanodrop4
  

• We performed restriction digest (SpeI, BSA, Buffer 4)of the products of the gel extraction
• We performed miniprep on 3 of the 4 overnight cultures (1k3 was not used due to minimal growth while 1A3, 1AT3 and 1C3 were able to grow sufficiently)
• We performed nanodrop quantification on the products of the miniprep
• nanodrop 5
  
• We performed restriction digest (SpeI, BSA, Buffer 4) of the products of the miniprep
• We made a second generation of culture of the plasmid carrying bacteria by injecting 20 uL of the original culture into 5 mL of fresh LB broth with the appropriate antibiotics.
• Using the generation 2 culture of the bacteria with the 1K3 plasmid, we mini prepped it
• We nanodrop quantified the mini prepped material
• nanodrop5
• We then ran restriction digest(SpeI, BSA, Buffer 4) of the mini prepped sample
• From the restriction digest of MS4-7 we performed PCR clean up
• We nano dropped the PCR clean up product.
• nanodrop 5
• We streaked plates from the overnight cultures of transformed bacteria (psB1A3, pSB1AT3, pSB1C3, pSB 1k3) and placed them into the 37C incubator.
• We prepared a presentation of our project for Wellesley college, whom we will be collaborating with during the summer :)
• We had a meeting with professor Doug and other undergrads from CIDAR lab.

• We worked on presentation for the Wellesley team.
• We performed PCR amplification with new sets of L0 primers
• DVL0_EF with DVL0_BR (MS8)
• DVL0_FF with DVL0_BR (MS9)
• DVL0_GF with DVL0_BR (MS10)
• DVL0_DF with DVL0_FR (MS11)
• DVL0_DF with DVL0_GR (MS12)
• DVL0_DF with DVL0_HR (MS13)
• We performed ligation of the successful amplified samples of MS4-7 and MF4
• MS4-7 with backbone of 1AT3
• MS4-7 with backbone of 1A3
• MF4 with backbone of 1K3
• We then performed gel electrophoresis of MS8-13, all were successful.
• gel 4
• We performed Gel extraction
• We nanodropped
• nanodrop6
  
• Attended meeting with Wellesley team.
• Ran 50 μL PCR reactions of MS8.1-13.1 for Restriction Digest
• Ran MgCl2 gradient of MF3 PCR to troubleshoot, along with DMSO separately.
• Performed Gel Electrophoresis of the PCR samples.
• We performed Gel Extraction
• gel 5
• We Nanodropped the samples
• nano7
• We performed Restriction digest with the samples MS9.1 and MS 12.1 that had enough DNA concentration
• We performed a new PCR in triplicate for the samples MS8.1, 10.1, 11.1 and 13.1 to achive a higher concentration of DNA
• We transformed the ligations with competent bacterial cells.
• MS4-7 with backbone of 1AT3
• MS4-7 with backbone of 1A3
• MF4 with backbone of 1K3

• We spread plated them with 20 μL of a 40 mg/mL X-Gal solution and 8 μL of a 0.5 IPTG solution for blue-white screening.
• We ran gel of MS8.1, 10.1, 13.1,11.1 to amplify more DNA template to achieve sufficient DNA concentration upon gel extraction
• gel6
• nano8
  
• We PCR clean up-ed MS9.1 and 12.1

• We found out that MS8-13 are all wrong due to primer dimer
• We redid the PCR of all level 0 primers that failed,
• FJ1 L0AB
• FJ2 L0EB
• FJ3 L0FB
• FJ4 L0GB
• FJ5 L0DF
• FJ6 L0DG
• FJ7 L0DH
• FJ8 L0DE
• FJ9 L0CD
• Instead of running a gel after the PCR, PCR clean up was performed
• The product of the clean up was then nanodropped:
• nano9
  

• Then the PCR clean up samples of FJ1-9 underwent restriction digest
• Ran gel of digest, bands came out too weak, but all present
• gel 7
• Redid digest with higher amount of DNA,
• new gel is much more stronger
• gel8
• performed gel extraction
• nanodropped gel extract
• nano10
  

• Performed PCR clean up on RD of 1A3, 1AT3, 1C3, 1K3 transformed at week1 and nanodropped

nano11

• Ligated 1K3 with pSMJSAF from samples of FJ1-9
• Transformed the ligations on to plates
• The plates did not yield positives for the transformation
• RD the backbones in two sets, one set with just SpeI other with both SpeI and EcoRI
• Gel of the RD showed that there were unexpected bands when cut with both enzymes, something not shown on the registry experience pages for the backbones.
• gel 9
• Plated bacteria transformed with the backbones
• Picked colonies from the plates
• Prepared plate media of Kan, Amp and CAM
• Miniprepped and nanodropped the cultures with the the backbones also made glycerol stocks of these.
• nano12
  
• We designed primers and gene synthesis template.
• Made glycerol stocks of 1AT3, 1A3, 1C3, 1K3 both for -20 and -80 celsius