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From 2011.igem.org

7/9 Saturday
• added DPN1 to RecA1 mutegensis
• incubated 4-6 hours
• PCR purfified
• transfromed RecA1:
o A3: RecA1 III from 7/7 T.C.: 5.0
o A5 : RecA1 V from 7/7 T.C.:5.2
o B3: : RecA1 III from 7/8 T.C: 5.0
o B5: : RecA1 V from 7/8 T.C. 5.0
• Transfomred test: T.C.: 2.2
• plated transformations and made freezer stock of RecA1III


7/10 Sunday
• got sequencing results back of RecA1 III and V. III is good even when blasting with NCBI,
• V is definetly reversed
• test did not grow
• ran gel of its PCR purification think had to much lyse because came out smeared just below 1000bp
• began digest of PCR purfication of part 2 from 7/8 miniprep of part 1 from ⅞
• litgate
• transformed
• plate


7/11Monday
• centrifuge broke
• nothing grew of test plates


7/12 Tuesday
• centrifuge fixed. Anisha miniprep RecaA1
• grew Part 1 and Part 2 from registry


7/13 Wednesday
• part cultures did not grow because I forgot to transform
• transformed from registry
o part 1 T.C.: 2.8
o Part 2 T.C.: 2.6
o cultured
• RecA1 mute results back--> mutation didn’t work
• New mutegeneiss PCR with extention time 2min 15 seconds to go overnight


7/14Thursday
• added DPN1 to RecA1 mute PCR, left to incubate
• transformed
o T.C.: 5.0
• plated
• mini prep part 1 and part 2
• PCR insert Part 2
• digested
• ligated
• transformed: T.C.: 2.2
• Byron started another PCR of RecA1 mute to run over night


7/15Friday
• colonies grew of test and RecA1 but very small so will let grow one more night and PCR colony the next day
• Byron
o added DPN1 and incubated for 6 hours
o heat killed at 80 degree fro 20 min (this should not have happened and probably why mutegensisi didn’t work)
o PCR purified product
o transformed
 T.C.: 2.0
o plated


7/16 Saturday
• colony PCR test: 2 from each plate 1A, 1B, 2A, 2B, 3A, 3B nothing grew on plate 4
• made culture of RecA1 mute 3 from one plate : 1A, 1B, 1C. Other plate only had one colony
• forgot to add oil before PCRing, started another PCR whil cultureing all sample


7/18
• mini-prepped RecA1 Ecor1 Pst1 mutegensis cultures A and B
• sent the test to sequencing. Terminator came out but the driver didn’t show up. Remu did not e
• going to assemble test again using B0015 mini-prep from 7/14 going to PCR driver from registry
• also going to try to assemble with Terminator as the insert and the driver as the vector. Terminator insert digest with E&S enzymes and Vector digest with E&X


7/19
• test using mini-prep from 7/14 of terminator and registry assembled with terminator as vector and driver as insert
• PCR driver
• Digest
• Ligate
• Transformed
o Time constant 2.0
• Plated
• Also assembled mini-prep terminator and mini-prep driver 7/14 but with terminator as insert and driver as insert
• PCR terminator
• Digest using buffer 4
• Ligate
• Transformed
o T.C. 2.0
• Plated A&B with assemble of terminator as insert and driver as vector
• Plated C&D with assemble of terminator as driver and driver as vector
RecA1 Mutagenesis
• Multisite mutagenesis was conducted for EcoR1 and Pst1
• Protocol followed was the desired mutagenesis from open wetware
• Also ran mutagenesis adjusting the original multisite muteagenesis protocol
o MgSO4: 0, 0.5, 1 ul
o DMSO: 0, 0.5, 1
o Without DMSO with primer
• Let PCR run overnight
Mutagenesis—multisite protocol varying MgSO4 and addition of Reverse Primer:
• With reverse primer
o 1: 0ul MgSO4
o 2: 0.5ul MgSO4
o 3: 1ul MgSO4
• Without reverse Primer
o 4: 0ul MgSO4
o 5: 0.5ul MgSO4
o 6: 1ul MgSO4
• Due to supplies was only able to do one mutagenesis, choose to do 4


7/20
• PCR colony tests
• Cultured test
RecA
• Incubated 4 hours
• PCR purified RecA1 mutagenesis
• Transformed
o T.C. 4.4
• Plated
• Reviewed sequencing results from 7/18, came back unsuccessful


7/21
• Mini-prep RecA1 send to sequencings
• Culture RecA1 mute1
• Mini-prep terminator and driver and RecA1 III
• Also made freezer stock
• PCR insert of terminator with extension time 45 seconds
• Plated two colonies (I, II) from transformation on 7/19 (PCR without reverse primer)
• Made culture


7/22
• No centrifuge tubes so unable to send to sequencing
• PCR purified terminator
• Digested terminator as insert and driver as vector
• Ligated
• Transformed
o T.C. 1.8
• Mini-prepped culture (I and II) from 7/21 and sent to sequencing
• Began other mutagenesis’s by beginning of PCR
o 1, 2, 3, 5, and 6


7/23
• Incubated Mutagenesis’s for 4 hours
• PCR purified
• Transformed
o T.C.
 1: 4.4
 2: 4.8
 3: 4.8
 5: 4.8
 6: 4.0


7/24
• Colonies were not very big waiting until Monday
• Began another RecA mutagenesis starting with PCR
o Primers Pst, Arg243, Lys 286
• Minipreped mutagenesis 4 and sent to sequencing


7/25
• Cultured colonies from test 1A, 1B, 2A, 2B, 3A, 3B, 5A, 5B, 6A, ^B and test
• Reviewed sequencing: unsuccessful
• Incubated second RecA mutagenesis
• PCR purified
• Transformed


7/26
• Minipreped colonies however was late to sequencing
• Began a third mutageneis with different amounts of DMSO and PCR annealing temperature


7/27
• Cultured test again
• Sequencing results had a lot of Ns
• Started new test assembly
o 1: Vector = Driver Enzymes E&X, Insert=Terminator, Enzymes E&S
o PCRed, ligated, transformed (T.C. 2.4) palted
• Incubated RecA
• PCR purfified
• Transformed (T.C: 5.2)
• plated


7/28
• Pst primer and EcoR1 primers were re-ordered because discovered they were incorrect
• No colonies grew on test plate
• Grew terminator and driver from freezer stock


7/29
• Cultured RecA from plates
• Mini-prepred terminator and driver and continued assembly (T.C. 3.2)
• Mini-prep RecA mutagenesis and sent to sequencing


7/30
• cultured test assembly
• reviewed RecA results: unsuccessful


7/31
• started new RecA mutagenesis
o varying concentrations and PCR temperatures
• mini-prep test and sent to sequencing


8/1
• continued RecA mutagenesis
• reviewed test sequencing


8/2
• reviewed test and RecA mini-preps using a nanodrop because suspicion of bad sequencing read
• several did not turn out well so re-mini-prepped and sent to sequencing


8/3
• checked sequencing still unsuccessful


8/4
• discovered mini-prep test kit was contaminated
• retrieved new kit and began mutagenesis


8/8
• Began test assembly
• Using terminator and driver from 7/18 (had be verified through sequencing that it was terminator and driver)
• Digest
o Driver as vector enzymes E&X
o Terminator as insert E&S
• Ligated
• Transformed ligation
o T.C.: 2.2
• Plated


8/9
• Cultured parts for new assembly
• Colony PCR product from 8/8


8/12
• Cultured test, terminator and driver


8/13
• Miniprepped from 8/12


9/8
- Using CBAR for mutagenesis
- Designed oligos -> ordered
- PCR’d boh B0015 and I763007


9/9
- Oligo arrived -> ran PCR
- Rec A Lys Mutation
o Pst Forward, EcoR1 Reverse
o Pst R, EcoR1 F
- RecA Pst Mutagenesis
o Arg F, Lys R
o Arg R, Lys F
- PCR Purified – B0015 and I763007
- Digested
o Term
 Vector = S, P
 Insert = E, S
o Driver
 Vector = E, X
 Insert = X, P
- Digest Purified Vectors
- Ligated
o Test 1 = Term (V) + Driver (I)
o Test 2 = Term (I) + Driver (V)
- Transformed
o Test 1 = 3.0
o Test 2 = 3.0
- Plated
o A plates


9/12
- PCR Purified
- Nanodropped
o 14.6 ng/µL
o 19.8
o 41.5
o 9.7
- Fentamole Calculations were done
- Waited to verify calculation with supervisor
- Diluted PCRs to correct concentration
- Combined the following together w/ CBAR mixture:
o Pst F + EcoR1 R with Pst R + EcoR1 F
o Arg F + Lys R with Arg R + Lys F
- Incubated at 50 degrees C for 1 hour.
- PCR purified
- Transformed .5 µL on K resistance
- Plated
- PCR Colony – Test 1 and Test 2
- Ran on gel
o Looked good
- Cultured
- Transformed 2nd half of circuit
o T.C. =5.2
- Cultured part


9/13
- Miniprepped test 1, 2, and the second half of the circuit (B)
- Sent to sequencing


9/14
- Colony stab and cultured
- Read sequencing results – Test 1, 2, B
o Test 1 = not good
o Test 2 = GOOD! :D
o Test B = GOOD! :D
o PCR’d all parts that worked


9/15
- CBAR Epic fail
- Ran PCR product on gel
o Looked good
- PCR Purified
- Digested
o Part 1 (Test 2)
 Vector = S,D
 Insert = E, S
o Part 2 (Test B)
 Vector = E, X
 Insert = X, P
- Ligated
o Test 1 = Part 1 (V) + Part 2 (I)
o Test 2 = Part 2 (V) + Part 1 (I)
- Transformed
o Test 1 = 2.8
o Test 2 = 2.6
- Plated
o A Resistence


9/16
- Test 1 nor Test 2 grew – started over
9/19
- Mutagenesis
o PE + Arg -> fail
o PE + Lys -> worked
o LE + Arg -> fail
o LE + Pst1 -> fail

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