SBB10Ntbk-Mesa

=Info= Profile Project Protocols =Parts Project=

sbb27 (KRM27)
sbb27: CA42 rep Protein PCR KRM001 and KRM002 on pEC22-CA42 (987 bp, EcoRI/BamHI) Sub into pBca9523-Bca1144           (EcoRI/BamHI, 2472+910, L) Product is pBca9523-sbb27         {rbs.repCA42!} KRM001  Forward oligo for cloning of rbs.repCA42! ccataGAATTCatgAGATCTcccggagagaatcgtaacac KRM002  Reverse oligo for cloning of rbs.repCA42! ctgatGGATCCctattttccgcttttccag

sbb16 (KRM16)
sbb16: phiC31 attP Wobble KRM003/KRM004           (115bp, EcoRI/BamHI) Sub into pBjk2741-Bca1144        (EcoRI/BamHI, 2170+910, L) Product is pBjk2741-sbb16     {phiC31 attP} KRM003  Forward construction of phiC31 attP basic part ccataGAATTCatgAGATCTagaagcggttttcgggagtagtgccccaactggggtaacctttgag KRM004  Reverse construction of phiC31 attP basic part ctgatGGATCCgtgtcatgtcggcgaccctacgcccccaactgagagaactcaaaggttaccccagttg

2/17/2010
Received Oligos: KRM001, KRM002, KRM003, KRM004 Diluted all oligos to a 100μM concentration

Made 10μM dilutions of KRM001 and KRM 002 9μl H2O 1μl 100μM oligo

PCR and Wobble Reaction
Prepared one wobble and one PCR reaction, KRM16 and KRM27 respectively. Wobble Reaction (KRM16) 29μl ddH2O 5μl 2 5μl dNTPs 5μl KRM003 (100μM) 5μl KRM004 (100μM) 0.75μl 1 PCR Reaction (KRM27) 24μl ddH2O 3.3μl 2 3.3μl dNTPs 1μl KRM003 (10μM) 1μl KRM004 (10μM) 0.5μl 1 0.5μl Template DNA (pEC22-CA42)

2/22/2010
Zymo Clean Up on PCR (KRM27) Elute in 33μl Small Fragment Zymo on Wobble (KRM16) Elute in 50μl

PCR and Wobble Analytical Gels
Ran an Analytical Gel for Purified PCR (KRM27) and Wobble (KRM16) samples 3μl Purified PCR (KRM27) 7μl Loading Buffer 3μl Purified Wobble (KRM16) 7μl Loading Buffer

Purified Samples were stored in140L Box A

Gel Results KRM27 expected size is 971bp (Second to far right lane) KRM16 expected size is 99bp (Far right lane)

EcoRI/BamHI Digests
PCR EcoRI/BamHI Digest on Purified PCR (KRM27) Remaining Purified PCR product in tube labeled->KRM27, Purified PCR product, 2/22/10 Wobble EcoRI/BamHI Digest on Purified Wobble (KRM16) All of Purified Wobble product was used in digest Small Fragment Zymo on Wobble Digest (KRM16 Digest) Elute in 50μl Wobble digest product in tube labeled->KRM16 Digest, Purified Digest, 2/24/10

Preparative and Analytical Gels for Digests
Ran a Preparative and an Analytical Gel for PCR Digest (KRM27 Digest) and Wobble Digest (KRM16 Digest) samples, respectively 9μl PCR Digest (KRM27 Digest) 1μl 6X Loading Buffer 3μl Wobble Digest (KRM16 Digest) 7μl Loading Buffer

Cut out PCR Digest from preparative gel, place in 1.5ml tube Add 600μl ADB buffer @ 55°C

Gel Results KRM16 Digest expected size is 99bp (Far right lane)



Samples were stored in 140L Box A

3/1/2010
Zymo Gel Purification on KRM27 Digest Elute in 33μl

Ligation Reactions
KRM27 Digest into pBca9523-Bca1144 KRM16 Digest into pBjk2741-Bca1144

Negative Control Ligations pBca9523-Bca1144 (no insert) pBjk2741-Bca1144 (no insert)

Transformation
KRM27/pBca9523 => KRM27 Digest into pBca9523-Bca1144 KRM16/pBjk2741 => KRM16 Digest into pBjk2741-Bca1144

Minipreps
Miniprep samples: sb0027: KRM27-1,KRM27-2,KRM27-3,KRM27-4 sb0016: KRM16-1,KRM16-2,KRM16-3,KRM16-4

Mapping
Miniprep Digestion with EcoRI/BamHI on all miniprep samples

Gel Results
KRM27-1,KRM27-2,KRM27-3,KRM27-4 expected insert size is 971bp KRM16-1,KRM16-2,KRM16-3,KRM16-4 expected insert size is 99bp


 * 1) Ladder
 * 2) sbb22 Digest
 * 3) sbb20 Digest
 * 4) KRM27 1
 * 5) KRM27 2
 * 6) KRM27 3
 * 7) KRM27 4
 * 8) KRM16 1
 * 9) KRM16 2
 * 10) KRM16 3
 * 11) KRM16 4
 * 12) FK114
 * 13) Ladder

Remapping of sb0016 Samples
Digested KRM16-1,KRM16-2,KRM16-3,KRM16-4 with BamHI/XhoI

Gel Results
KRM16-1,KRM16-2,KRM16-3,KRM16-4 expected insert size is 783bp All samples had an insert at the proper band size. Gel Pic

Sequencing
KRM16-1,KRM16-2 and KRM27-1,KRM-2 were chosen to be sequenced. Samples were given to Chris and were loaded as [Plate A:C1],[Plate B:C1] and [Plate A:D1],[Plate B:D1], respectively.

Sequence of sbb16
Sequence for [Plate A:C1] is a perfect read. Construct is pBjk2471-sbb16. See: Sequencing Log.

Sequence of sbb27
Sequence for [Plate A:D1] has a silent mutation (349bp A->G) Ala->Ala, but part is still good. Construct is pBca9523-sbb27. See: Sequencing Log.

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